Anti-IL-22R antibodies

ABSTRACT

The present invention relates to antibodies and antigen binding fragments thereof which bind to the cytokine receptor IL-22R, particularly human IL-22R. The invention also relates to pharmaceutical compositions comprising said antibodies or antigen binding fragments thereof, and methods of treating psoriasis, psoriatic arthritis or atopic dermatitis.

RELATED APPLICATIONS

This application is a continuation of International Patent ApplicationNo. PCT/EP2017/067923, filed Jul. 14, 2017, which claims priority toGreat Britain Patent Application No. 1612337.4, filed Jul. 15, 2016, theentire disclosures of which are hereby incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Nov. 12, 2018, isnamed 597365_AGX5-034PCCON_Sequence_Listing.txt and is 63,096 bytes insize.

FIELD OF THE INVENTION

The present invention relates to antibodies and antigen bindingfragments thereof which bind to the cytokine receptor IL-22R,particularly human IL-22R. The IL-22R antibodies and antigen bindingfragments of the invention exhibit distinct properties, particularlydistinct combinations of properties, as compared with IL-22R antibodiesdescribed in the prior art.

BACKGROUND TO THE INVENTION

IL-22R (also known as IL-22R1 and IL-22RA) is a type II cytokinereceptor selectively expressed on skin and epithelial cells. Thisreceptor mediates signalling via three cytokines: interleukin 22(IL-22), interleukin 20 (IL-20) and interleukin 24 (IL-24). Cytokinesignalling via the IL-22R requires the formation of heterodimericcomplexes at the cell surface. As shown in FIG. 1, IL-22 binds to andsignals via a complex consisting of IL-22R and IL-10Rβ (also known asIL-10R2), whereas IL-20 and IL-24 bind to and signal via a heterodimericcomplex consisting of IL-22R and IL-20Rβ (also known as IL-20R2).

Interleukin-22 is a cytokine expressed by immune cells, particularlyactivated dendritic cells and T cells. Once produced by the immunesystem, IL-22 exerts its biological effects by binding to and activatingIL-22R on epithelial cells. Activation of the IL-22R-IL-10Rβ complexdownstream of IL-22 binding leads to pro-inflammatory responses, theinduction of anti-microbial proteins that are critical for host defenseagainst bacterial pathogens, and protective effects in some organs suchas the lungs and liver. IL-22 has also been implicated in diseasepathology, particularly in the development of inflammatory disorderssuch as psoriasis, psoriatic arthritis and atopic dermatitis (Ma et al.J Clin. Invest. 118: 597-607 (2008); Van Belle et al. J Immunol. January1; 188(1):462-9 (2012); Sabat et al. Nat. Rev. Drug Discov. 13(1): 21-38(2014)).

The crystal structure of IL-22 in complex with the extracellular domainof IL-22R has been solved, and has provided important insights into howthis ligand associates with its receptor (Jones et al. Structure 16(9):1333-1344 (2008)). The extracellular region of IL-22R includes twofibronectin type III (FBNIII) domains (D1 and D2) oriented atapproximately right angles to one another. Five loops located at theinterface of these domains are primarily responsible for engaging IL-22residues in the ligand-receptor complex. The IL-22 residues thatcontribute to receptor binding are clustered at two sites in the ligand,site 1a and site 1b. Insights into the critical residues contributed byboth the receptor and the ligand have revealed ways in which thisinteraction could be disrupted to abrogate IL-22 signalling as atherapeutic strategy.

Interleukin-20 and interleukin-24 are expressed by monocytes andkeratinocytes and similar to IL-22, these cytokines have been found toplay a role in skin homeostasis and pathology. It follows, thatstrategies to inhibit or reduce signalling downstream of IL-22R byblocking the binding of ligands that activate this receptor could havetherapeutic utility, particularly in the treatment of skin conditionssuch as psoriasis and atopic dermatitis.

Antibodies that bind to IL-22R and block the interaction between IL-22and IL-22R have been developed. For example, WO2011/061119 describes ahumanised IL-22R antibody produced from a mouse anti-human monoclonalantibody originally described in WO2006/047249. This humanised antibody,which will be referred to herein as “280-346-TSY” was shown to inhibitIL-22 signalling via IL-22RA in a cell proliferation assay, andinhibited IL-23-induced ear inflammation in a mouse model of psoriasis.

SUMMARY OF INVENTION

The present invention improves upon the state of the art by providingantibodies, or antigen binding fragments thereof, which bind to thecytokine receptor IL-22R, and exhibit properties that are different toIL-22R antibodies described in the prior art. The antibodies or antigenbinding fragments thereof typically exhibit combinations of propertiesthat are distinct and in certain cases superior, to the properties ofthe prior art IL-22R antibodies, particularly the humanised IL-22Rantibody described in WO2011/061119. The properties of these antibodiescan be particularly advantageous with regard to use in human therapy,particularly for the treatment of conditions such as psoriasis,psoriatic arthritis and atopic dermatitis.

In a first aspect, the present invention provides an antibody, or anantigen binding fragment thereof, which binds to human IL-22R, whereinthe antibody or antigen binding fragment thereof binds to an epitopewithin the IL-22R protein that does not include Tyr60.

In certain embodiments, the antibodies or antigen binding fragmentsthereof possess one or more additional properties selected from thefollowing:

-   -   (i) the ability to bind an epitope of human IL-22R located at        least in part in the D2 domain of the IL-22R protein;    -   (ii) the ability to bind human IL-22R with high affinity;    -   (iii) the ability to block binding of IL-22 to human IL-22R;    -   (iv) the ability to inhibit IL-22 dependent activation of        IL-22R;    -   (v) the ability to inhibit IL-20 dependent activation of IL-22R;    -   (vi) the ability to inhibit IL-22 and IL-20 dependent activation        of IL-22R; and    -   (vii) lack of cross-reactivity with murine IL-22R.

The antibodies or antigen binding fragments may exhibit high humanhomology, as defined elsewhere herein. In certain embodiments, theantibodies or antigen binding fragments thereof comprise a heavy chainvariable domain (VH) and a light chain variable domain (VL) wherein theVH and/or VL domains or one or more complementarity determining regions(CDRs) thereof are derived from an animal of the Camelidae family i.e.are camelid-derived. The antibodies or antigen binding fragmentsexhibiting high human homology or having at least one camelid-derivedCDR sequence, VH and/or VL domain may be humanised or germlined variantsof VH or VL domains from camelid conventional antibodies, wherein theterms “humanised” and “germlined” are as defined elsewhere herein.

In non-limiting embodiments the invention provides the followingantibodies, or antigen binding fragments thereof, which are defined byreference to specific structural characteristics, i.e. specified aminoacid sequences of either the CDRs (one or more of SEQ ID NOs: 2, 4, 6,9, 11, 13, 34, 36, 41, 43 (heavy chain CDRs) or SEQ ID NOs: 16, 18, 20,23, 25, 27, 47, 54, 57, 59 (light chain CDRs)) or entire variabledomains (one or more of SEQ ID NOs: 29, 31, 63, 65 (heavy chain variabledomains) or SEQ ID NOs: 30, 32, 62, 64, 66 (light chain variabledomains)). All of these antibodies bind to the human cytokine receptorIL-22R.

In particular embodiments, the antibodies defined by the followingstructural characteristics may exhibit high human homology, as definedherein. The antibodies may be monoclonal antibodies produced byrecombinant means. The CDRs of the following IL-22R antibodies may becamelid-derived, i.e. derived from conventional antibodies raised byimmunisation of camelids (specifically llama). The invention alsoprovides humanised or human germlined variants, affinity variants andvariants containing conservative amino acid substitutions, as definedherein.

Embodiments of the IL-22R antibodies of the invention are now furtherdescribed by reference to structural characteristics.

In one embodiment, there is provided an antibody or antigen bindingfragment thereof, which binds to the cytokine receptor IL-22R, saidantibody or antigen binding fragment comprising a heavy chain variabledomain (VH) comprising a heavy chain CDR3 selected from:

-   -   SEQ ID NO: 6 [VGFSGTYYSES], or sequence variant thereof    -   SEQ ID NO: 13 [PPGPFKAHYNGMKY], or sequence variant thereof,    -   SEQ ID NO: 43 [PPGPFKAHYNGAKY], or sequence variant thereof,        wherein the sequence variant comprises one, two or three amino        acid substitutions (e.g. conservative substitutions, humanising        substitutions or affinity variants) in the recited sequence.

The heavy chain variable domain of the antibody or antigen bindingfragment thereof may alternatively or in addition comprise a heavy chainCDR2 selected from:

-   -   SEQ ID NO: 4 [SIYNDGSNTAYSDSVKG], or sequence variant thereof,    -   SEQ ID NO: 11 [GIHISGGITYYLDSVKG], or sequence variant thereof,    -   SEQ ID NO: 36 [SIYNDASNTAYSDSVKG], or sequence variant thereof,    -   SEQ ID NO: 41 [GIHISGGITYYTDSVKG], or sequence variant thereof,        wherein the sequence variant comprises one, two or three amino        acid substitutions (e.g. conservative substitutions, humanising        substitutions or affinity variants) in the recited sequence.

The heavy chain variable domain of the antibody or antigen bindingfragment thereof may alternatively or in addition comprise a heavy chainCDR1 selected from:

-   -   SEQ ID NO: 2 [SYDMS], or sequence variant thereof,    -   SEQ ID NO: 9 [SYFMS], or sequence variant thereof,    -   SEQ ID NO: 34 [SYDMN], or sequence variant thereof,        wherein the sequence variant comprises one, two or three amino        acid substitutions (e.g. conservative substitutions, humanising        substitutions or affinity variants) in the recited sequence.

Alternatively or in addition, the antibodies or antigen binding fragmentthereof, which bind to the cytokine receptor IL-22R, may comprise alight chain variable domain (VL) comprising a light chain CDR3 selectedfrom:

-   -   SEQ ID NO: 20 [QSGSSSANAV], or sequence variant thereof,    -   SEQ ID NO: 27 [ASYRLYADYV], or sequence variant thereof,    -   SEQ ID NO: 54 [QSGSSSSNAV], or sequence variant thereof,        wherein the sequence variant comprises one, two or three amino        acid substitutions (e.g. conservative substitutions, humanising        substitutions or affinity variants) in the recited sequence.

The light chain variable domain of the antibody or antigen bindingfragment thereof may alternatively or in addition comprise a light chainCDR2 selected from:

-   -   SEQ ID NO: 18 [GNNNRPS], or sequence variant thereof    -   SEQ ID NO: 25 [KVNTRSS], or sequence variant thereof,    -   SEQ ID NO: 47 [GQNNRPS], or sequence variant thereof,    -   SEQ ID NO: 59 [EVNKRSS], or sequence variant thereof,        wherein the sequence variant comprises one, two or three amino        acid substitutions (e.g. conservative substitutions, humanising        substitutions or affinity variants) in the recited sequence.

The light chain variable domain of the antibody or antigen bindingfragment thereof may alternatively or in addition comprise a light chainCDR1 selected from:

-   -   SEQ ID NO: 16 [QGGYYAH], or sequence variant thereof    -   SEQ ID NO: 23 [TGTSRDIGDYNYVS], or sequence variant thereof,    -   SEQ ID NO: 57 [TGTSSDIGSYNYVS], or sequence variant thereof,        wherein the sequence variant comprises one, two or three amino        acid substitutions (e.g. conservative substitutions, humanising        substitutions or affinity variants) in the recited sequence.

In certain embodiments, there is provided an antibody or antigen bindingfragment thereof, which binds to the cytokine receptor IL-22R, theantibody or antigen binding fragment thereof comprising a combination ofvariable heavy chain CDR3 (HCDR3), variable heavy chain CDR2 (HCDR2) andvariable heavy chain CDR1 (HCDR1) wherein the combination is selectedfrom the group consisting of:

(i) HCDR3 comprising SEQ ID NO: 6; HCDR2 comprising SEQ ID NO: 36; HCDR1comprising SEQ ID NO: 34;

(ii) HCDR3 comprising SEQ ID NO: 43; HCDR2 comprising SEQ ID NO: 41;HCDR1 comprising SEQ ID NO: 9;

(iii) HCDR3 comprising SEQ ID NO: 6; HCDR2 comprising SEQ ID NO: 4;HCDR1 comprising SEQ ID NO: 2; and

(iv) HCDR3 comprising SEQ ID NO: 13; HCDR2 comprising SEQ ID NO: 11;HCDR1 comprising SEQ ID NO: 9.

Alternatively or in addition, the antibodies or antigen binding fragmentthereof, which bind to the cytokine receptor IL-22R may comprise acombination of variable light chain CDR3 (LCDR3), variable light chainCDR2 (LCDR2) and variable light chain CDR1 (LCDR1) selected from thegroup consisting of:

(i) LCDR3 comprising SEQ ID NO: 54; LCDR2 comprising SEQ ID NO: 47;LCDR1 comprising SEQ ID NO: 16;

(ii) LCDR3 comprising SEQ ID NO: 27; LCDR2 comprising SEQ ID NO: 59;LCDR1 comprising SEQ ID NO: 57;

(iii) LCDR3 comprising SEQ ID NO: 20; LCDR2 comprising SEQ ID NO: 47;LCDR1 comprising SEQ ID NO: 16;

(iv) LCDR3 comprising SEQ ID NO: 20; LCDR2 comprising SEQ ID NO: 18;LCDR1 comprising SEQ ID NO: 16; and

(v) LCDR3 comprising SEQ ID NO: 27; LCDR2 comprising SEQ ID NO: 25;LCDR1 comprising SEQ ID NO: 23.

In certain embodiments, provided herein are antibodies or antigenbinding fragments thereof, which bind to the cytokine receptor IL-22R,wherein the antibodies or antigen binding fragments comprise acombination of variable heavy chain CDR3 (HCDR3), variable heavy chainCDR2 (HCDR2) and variable heavy chain CDR1 (HCDR1), variable light chainCDR3 (LCDR3), variable light chain CDR2 (LCDR2) and variable light chainCDR1 (LCDR1) according to the embodiments described below.

In one embodiment, provided herein is an antibody or antigen bindingfragment thereof, which binds to the cytokine receptor IL-22R andcomprises the combination of VH and VL CDR sequences: HCDR3 comprisingSEQ ID NO: 6; HCDR2 comprising SEQ ID NO: 36; HCDR1 comprising SEQ IDNO: 34; LCDR3 comprising SEQ ID NO: 54; LCDR2 comprising SEQ ID NO: 47;and LCDR1 comprising SEQ ID NO: 16.

In one embodiment, provided herein is an antibody or antigen bindingfragment thereof, which binds to the cytokine receptor IL-22R andcomprises the combination of VH and VL CDR sequences: HCDR3 comprisingSEQ ID NO: 43; HCDR2 comprising SEQ ID NO: 41; HCDR1 comprising SEQ IDNO: 9; LCDR3 comprising SEQ ID NO: 27; LCDR2 comprising SEQ ID NO: 59;and LCDR1 comprising SEQ ID NO: 57.

In one embodiment, provided herein is an antibody or antigen bindingfragment thereof, which binds to the cytokine receptor IL-22R andcomprises the combination of VH and VL CDR sequences: HCDR3 comprisingSEQ ID NO: 6; HCDR2 comprising SEQ ID NO: 4; HCDR1 comprising SEQ ID NO:2; LCDR3 comprising SEQ ID NO: 20; LCDR2 comprising SEQ ID NO: 47; andLCDR1 comprising SEQ ID NO: 16.

In one embodiment, provided herein is an antibody or antigen bindingfragment thereof, which binds to the cytokine receptor IL-22R andcomprises the combination of VH and VL CDR sequences: HCDR3 comprisingSEQ ID NO: 6; HCDR2 comprising SEQ ID NO: 4; HCDR1 comprising SEQ ID NO:2; LCDR3 comprising SEQ ID NO: 20; LCDR2 comprising SEQ ID NO: 18; andLCDR1 comprising SEQ ID NO: 16.

In one embodiment, provided herein is an antibody or antigen bindingfragment thereof, which binds to the cytokine receptor IL-22R andcomprises the combination of VH and VL CDR sequences: HCDR3 comprisingSEQ ID NO: 13; HCDR2 comprising SEQ ID NO: 11; HCDR1 comprising SEQ IDNO: 9; LCDR3 comprising SEQ ID NO: 27; LCDR2 comprising SEQ ID NO: 25;and LCDR1 comprising SEQ ID NO: 23.

In certain embodiments, provided herein are antibodies or antigenbinding fragments thereof, which bind to the cytokine receptor IL-22R,wherein the antibodies or antigen binding fragments comprise a heavychain variable domain (VH) selected from the following:

-   -   (i) a VH comprising or consisting of the amino acid sequence of        SEQ ID NO: 29 or 31    -   (ii) an affinity variant or human germlined variant of a VH        comprising or consisting of the amino acid sequence of SEQ ID        NO: 29 or 31; or    -   (iii) a VH comprising or consisting of an amino acid sequence        having at least 80%, at least 85%, at least 90%, at least 95%,        at least 97%, at least 98%, at least 99% identity to the amino        acid sequence of SEQ ID NO: 29 or 31.

Alternatively or in addition, the antibodies or antigen bindingfragments may comprise a light chain variable domain (VL) selected fromthe following:

-   -   (i) a VL comprising or consisting of the amino acid sequence of        SEQ ID NO: 30, 32 or 62    -   (ii) an affinity variant or human germlined variant of a VH        comprising or consisting of the amino acid sequence of SEQ ID        NO: 30, 32 or 62; or    -   (iii) a VL comprising or consisting of an amino acid sequence        having at least 80%, at least 85%, at least 90%, at least 95%,        at least 97%, at least 98%, at least 99% identity to the amino        acid sequence of SEQ ID NO: 30, 32 or 62.

For embodiments wherein the domains of the antibodies or antigen bindingfragments are defined by a particular percentage sequence identity to areference sequence, the VH and/or VL domains may retain identical CDRsequences to those present in the reference sequence such that thevariation is present only within the framework regions.

In certain embodiments, the antibodies of the invention may include theCH1 domain, hinge region, CH2 domain and CH3 domain of a human antibody,in particular human IgG1, IgG2, IgG3 or IgG4.

Particularly preferred antibodies of the present invention are describedbelow.

230C9 and Antibodies Related Thereto

In certain embodiments, there is provided an isolated antibody, orantigen binding fragment thereof, which specifically binds IL-22R, saidantibody or antigen binding fragment comprising a heavy chain variabledomain wherein:

the variable heavy chain CDR3 sequence is SEQ ID NO:6 [VGFSGTYYSES] orsequence variant thereof;

the variable heavy chain CDR2 sequence is SEQ ID NO:36[SIYNDASNTAYSDSVKG] or sequence variant thereof; and

the variable heavy chain CDR1 sequence is SEQ ID NO:34 [SYDMN] orsequence variant thereof, and

wherein the sequence variant comprises one, two or three amino acidsubstitutions (e.g., conservative substitutions, humanisingsubstitutions or affinity variants) in the recited sequence.

The antibody or antigen binding fragment may further comprise a lightchain variable domain wherein:

the variable light chain CDR3 sequence is SEQ ID NO:54 [QSGSSSSNAV] orsequence variant thereof;

the variable light chain CDR2 sequence is SEQ ID NO:47 [GQNNRPS] orsequence variant thereof; and

the variable light chain CDR1 sequence is SEQ ID NO:16 [QGGYYAH] orsequence variant thereof, and

wherein the sequence variant comprises one, two or three amino acidsubstitutions (e.g., conservative substitutions, humanisingsubstitutions or affinity variants) in the recited sequence.

In certain embodiments, there is provided an isolated antibody, orantigen binding fragment thereof, which specifically binds IL-22R, saidantibody or antigen binding fragment comprising a heavy chain variabledomain wherein:

the variable heavy chain CDR3 sequence comprises or consists of SEQ IDNO:6 [VGFSGTYYSES];

the variable heavy chain CDR2 sequence comprises or consists of SEQ IDNO:36 [SIYNDASNTAYSDSVKG];

the variable heavy chain CDR1 sequence comprises or consists of SEQ IDNO:34 [SYDMN];

the variable light chain CDR3 sequence comprises or consists of SEQ IDNO:54 [QSGSSSSNAV];

the variable light chain CDR2 sequence comprises or consists of SEQ IDNO:47 [GQNNRPS]; and

the variable light chain CDR1 sequence comprises or consists of SEQ IDNO:16 [QGGYYAH].

The antibodies or antigen binding fragments thereof may comprise a heavychain variable domain (VH) comprising the amino acid sequence of SEQ IDNO: 63 and optionally a light chain variable domain (VL) comprising theamino acid sequence of SEQ ID NO: 64. In certain embodiments, providedherein are monoclonal antibodies or antigen binding fragments thereof,comprising a heavy chain variable domain and a light chain variabledomain, the heavy chain variable domain comprising a VH sequence with atleast 85% sequence identity, or at least 90% sequence identity, or atleast 95% sequence identity, or at least 97%, 98% or 99% sequenceidentity, to the amino acid sequence shown as SEQ ID NO:63 and/or thelight chain variable domain comprising a VL with at least 85% sequenceidentity, or at least 90% sequence identity, or at least 95% sequenceidentity, or at least 97%, 98% or 99% sequence identity, to the aminoacid sequence shown as SEQ ID NO:64. For embodiments wherein the domainsof the antibodies or antigen binding fragments are defined by aparticular percentage sequence identity to a reference sequence, the VHand/or VL domains may retain identical CDR sequences to those present inthe reference sequence such that the variation is present only withinthe framework regions. In certain embodiments, the antibodies or antigenbinding fragments comprising heavy chain variable domains and/or lightchain variable domains defined as having a particular percentageidentity to SEQ ID NOs: 63 and 64, respectively will have the followingCDR sequences:

a variable heavy chain CDR3 sequence comprising or consisting of SEQ IDNO:6 [VGFSGTYYSES];

a variable heavy chain CDR2 sequence comprising or consisting of SEQ IDNO:36 [SIYNDASNTAYSDSVKG];

a variable heavy chain CDR1 sequence comprising or consisting of SEQ IDNO:34 [SYDMN];

a variable light chain CDR3 sequence comprising or consisting of SEQ IDNO:54 [QSGSSSSNAV];

a variable light chain CDR2 sequence comprising or consisting of SEQ IDNO:47 [GQNNRPS]; and

a variable light chain CDR1 sequence comprising or consisting of SEQ IDNO:16 [QGGYYAH].

The antibodies which specifically bind IL-22R may comprise at least onefull-length immunoglobulin heavy chain and/or at least one full-lengthlambda or kappa light chain. In certain embodiments, the antibodiescomprise a heavy chain comprising the amino acid sequence of SEQ ID NO:67 and a light chain comprising the amino acid sequence of SEQ ID NO:68. In certain embodiments, provided herein are monoclonal antibodiescomprising a heavy chain with at least 85% sequence identity, or atleast 90% sequence identity, or at least 95% sequence identity, or atleast 97%, 98% or 99% sequence identity, to the amino acid sequenceshown as SEQ ID NO:67 and/or a light chain with at least 85% sequenceidentity, or at least 90% sequence identity, or at least 95% sequenceidentity, or at least 97%, 98% or 99% sequence identity, to the aminoacid sequence shown as SEQ ID NO:68. For embodiments wherein the chainsof the antibodies are defined by a particular percentage sequenceidentity to a reference sequence, the heavy chain and/or light chain mayretain identical CDR sequences to those present in the referencesequence such that the variation is present only outside the CDRregions.

223G5 and Antibodies Related Thereto

In certain embodiments, there is provided an isolated antibody, orantigen binding fragment thereof, which specifically binds IL-22R, saidantibody or antigen binding fragment comprising a heavy chain variabledomain wherein:

the variable heavy chain CDR3 sequence is SEQ ID NO:43 [PPGPFKAHYNGAKY]or sequence variant thereof;

the variable heavy chain CDR2 sequence is SEQ ID NO:41[GIHISGGITYYTDSVKG] or sequence variant thereof; and

the variable heavy chain CDR1 sequence is SEQ ID NO:9 [SYFMS] orsequence variant thereof, and

wherein the sequence variant comprises one, two or three amino acidsubstitutions (e.g., conservative substitutions, humanisingsubstitutions or affinity variants) in the recited sequence.

The antibody or antigen binding fragment may further comprise a lightchain variable domain wherein:

the variable light chain CDR3 sequence is SEQ ID NO:27 [ASYRLYADYV] orsequence variant thereof;

the variable light chain CDR2 sequence is SEQ ID NO:59 [EVNKRSS] orsequence variant thereof; and

the variable light chain CDR1 sequence is SEQ ID NO:57 [TGTSSDIGSYNYVS]or sequence variant thereof, and

wherein the sequence variant comprises one, two or three amino acidsubstitutions (e.g., conservative substitutions, humanisingsubstitutions or affinity variants) in the recited sequence.

In certain embodiments, there is provided an isolated antibody, orantigen binding fragment thereof, which specifically binds IL-22R, saidantibody or antigen binding fragment comprising a heavy chain variabledomain wherein:

the variable heavy chain CDR3 sequence comprises or consists of SEQ IDNO:43 [PPGPFKAHYNGAKY];

the variable heavy chain CDR2 sequence comprises or consists of SEQ IDNO:41 [GIHISGGITYYTDSVKG];

the variable heavy chain CDR1 sequence comprises or consists of SEQ IDNO:9 [SYFMS];

the variable light chain CDR3 sequence comprises or consists of SEQ IDNO:27 [ASYRLYADYV];

the variable light chain CDR2 sequence comprises or consists of SEQ IDNO:59 [EVNKRSS]; and

the variable light chain CDR1 sequence comprises or consists of SEQ IDNO:57 [TGTSSDIGSYNYVS].

The antibodies or antigen binding fragments thereof may comprise a heavychain variable domain (VH) comprising the amino acid sequence of SEQ IDNO: 65 and optionally a light chain variable domain (VL) comprising theamino acid sequence of SEQ ID NO: 66. In certain embodiments, providedherein are monoclonal antibodies or antigen binding fragments thereof,comprising a heavy chain variable domain and a light chain variabledomain, the heavy chain variable domain comprising a VH sequence with atleast 85% sequence identity, or at least 90% sequence identity, or atleast 95% sequence identity, or at least 97%, 98% or 99% sequenceidentity, to the amino acid sequence shown as SEQ ID NO:65 and/or thelight chain variable domain comprising a VL with at least 85% sequenceidentity, or at least 90% sequence identity, or at least 95% sequenceidentity, or at least 97%, 98% or 99% sequence identity, to the aminoacid sequence shown as SEQ ID NO:66. For embodiments wherein the domainsof the antibodies or antigen binding fragments are defined by aparticular percentage sequence identity to a reference sequence, the VHand/or VL domains may retain identical CDR sequences to those present inthe reference sequence such that the variation is present only withinthe framework regions. In certain embodiments, the antibodies or antigenbinding fragments comprising heavy chain variable domains and/or lightchain variable domains defined as having a particular percentageidentity to SEQ ID NOs: 65 and 66, respectively will have the followingCDR sequences:

a variable heavy chain CDR3 sequence comprising or consisting of SEQ IDNO:43 [PPGPFKAHYNGAKY];

a variable heavy chain CDR2 sequence comprising or consisting of SEQ IDNO:41 [GIHISGGITYYTDSVKG];

a variable heavy chain CDR1 sequence comprising or consisting of SEQ IDNO:9 [SYFMS];

a variable light chain CDR3 sequence comprising or consisting of SEQ IDNO:27 [ASYRLYADYV];

a variable light chain CDR2 sequence comprising or consisting of SEQ IDNO:59 [EVNKRSS]; and

a variable light chain CDR1 sequence comprising or consisting of SEQ IDNO:57 [TGTSSDIGSYNYVS].

The antibodies which specifically bind IL-22R may comprise at least onefull-length immunoglobulin heavy chain and/or at least one full-lengthlambda or kappa light chain. In certain embodiments, the antibodiescomprise a heavy chain comprising the amino acid sequence of SEQ ID NO:69 and a light chain comprising the amino acid sequence of SEQ ID NO:70. In certain embodiments, provided herein are monoclonal antibodiescomprising a heavy chain with at least 85% sequence identity, or atleast 90% sequence identity, or at least 95% sequence identity, or atleast 97%, 98% or 99% sequence identity, to the amino acid sequenceshown as SEQ ID NO:69 and/or a light chain with at least 85% sequenceidentity, or at least 90% sequence identity, or at least 95% sequenceidentity, or at least 97%, 98% or 99% sequence identity, to the aminoacid sequence shown as SEQ ID NO:70. For embodiments wherein the chainsof the antibodies are defined by a particular percentage sequenceidentity to a reference sequence, the heavy chain and/or light chain mayretain identical CDR sequences to those present in the referencesequence such that the variation is present only outside the CDRregions.

Where particular antibodies, or antigen-binding regions, are identifiedas comprising a combination of a VH domain or heavy chain, defined byreference to a specific amino acid sequence, and a VL domain or a lightchain, also defined by reference to a specific amino acid sequence, thenfor each specific VH/VL or heavy chain/light chain combination listed(unless otherwise stated) this definition may be taken to includeantibodies, or antigen binding regions, formed by combination of a VHdomain/heavy chain having at least 85%, at least 90%, at least 95%, atleast 97%, or at least 99% sequence identity to the stated VH/heavychain amino acid sequence and a VL domain/light chain having at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least97%, or at least 99% sequence identity to the stated VL/light chainamino acid sequence. In each case the domains/chains defined by %sequence identity to the stated domain/chain amino acid sequences mayretain identical CDR sequences to those present in the stated VH/VLdomain or heavy/light chain amino acid sequences, whilst exhibitingamino acid sequence variation within the framework regions or otherregions outside the CDR regions.

Unless otherwise stated in the present application, % sequence identitybetween two amino acid sequences may be determined by comparing thesetwo sequences aligned in an optimum manner and in which the amino acidsequence to be compared can comprise additions or deletions with respectto the reference sequence for an optimum alignment between these twosequences. The percentage of identity is calculated by determining thenumber of identical positions for which the amino acid residue isidentical between the two sequences, by dividing this number ofidentical positions by the total number of positions in the comparisonwindow and by multiplying the result obtained by 100 in order to obtainthe percentage of identity between these two sequences. For example, itis possible to use the BLAST program, “BLAST 2 sequences” (Tatusova etal, “Blast 2 sequences—a new tool for comparing protein and nucleotidesequences”, FEMS Microbiol Lett. 174:247-250) available on the sitehttp://www.ncbi.nlm.nih.gov/gorf/bl2.html, the parameters used beingthose given by default (in particular for the parameters “open gappenalty”: 5, and “extension gap penalty”: 2; the matrix chosen being,for example, the matrix “BLOSUM 62” proposed by the program), thepercentage of identity between the two sequences to be compared beingcalculated directly by the program.

The IL-22R antibodies or antigen binding fragments thereof providedherein may each exhibit one, or any combination, of the followingproperties/features:

-   -   the antibody or antigen binding fragment may bind to an epitope        within the human IL-22R protein that does not include Tyr60;    -   the antibody or antigen binding fragment may bind to an epitope        located at least in part in the D2 domain of the human IL-22R        protein, wherein the D2 domain is amino acid residues 125 to 228        of SEQ ID NO: 71;    -   the antibody or antigen binding fragment may bind to human        IL-22R with high affinity;    -   the antibody or antigen binding fragment may block binding of        IL-22 to IL-22R;    -   the antibody or antigen binding fragment may inhibit        IL-22-dependent activation of IL-22R;    -   the antibody or antigen binding fragment may inhibit        IL-20-dependent activation of IL-22R;    -   the antibody or antigen binding fragment may inhibit        IL-22-dependent activation of IL-22R and IL-20-dependent        activation of IL-22R;    -   the antibody or antigen binding fragment may not cross-react        with murine IL-22R.

The IL-22R antibodies or antigen binding fragments thereof providedherein preferably exhibit two or more of the followingproperties/features:

-   -   the antibody or antigen binding fragment binds to an epitope        within human IL-22R protein that does not include Tyr60;    -   the antibody or antigen binding fragment binds to human IL-22R        with high affinity;    -   the antibody or antigen binding fragment inhibits        IL-22-dependent activation of IL-22R and IL-20-dependent        activation of IL-22R.

The IL-22R antibodies, or antigen binding fragments thereof, providedherein may be chimeric antibodies. In certain embodiments, theantibodies or antigen binding fragments thereof contain the hingeregion, CH2 domain and/or CH3 domain of a human IgG. In certainembodiments, the antibodies or antigen binding fragments thereof exhibithigh homology to a human IgG, preferably a human IgG1, wherein “highhuman homology” is as defined elsewhere herein. In certain embodiments,the antibodies or antigen binding fragments thereof comprise a heavychain variable domain (VH) and a light chain variable domain (VL)wherein the VH and/or VL domains or one or more complementaritydetermining regions (CDRs) thereof are derived from an animal of theCamelidae family i.e. are camelid-derived, preferably wherein thecamelid is a llama.

In further aspects, the invention also provides polynucleotide moleculeswhich encode the above-listed antibodies and antigen binding fragments,in addition to expression vectors comprising the polynucleotides, hostcells containing the vectors and methods of recombinantexpression/production of the antibodies described herein.

In a still further aspect, the invention provides a pharmaceuticalcomposition comprising any one of the IL-22R antibodies or antigenbinding fragments thereof described herein, and a pharmaceuticallyacceptable carrier or excipient.

A still further aspect of the invention concerns methods of medicaltreatment using the above-listed IL-22R antibodies or antigen bindingfragments thereof, particularly in the prophylaxis and/or treatment ofconditions such as psoriasis, psoriatic arthritis and atopic dermatitis.

These and other embodiments of the invention will be better appreciatedand understood when considered in conjunction with the followingdescription and the accompanying drawings. It should be understood,however, that the following description, while indicating variousembodiments of the invention and numerous specific details thereof, isgiven by way of illustration and not of limitation. Many substitutions,modifications, additions and/or rearrangements may be made within thescope of the invention without departing from the spirit thereof, andthe invention includes all such substitutions, modifications, additionsand/or rearrangements.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the different receptor complexes that mediate signallingvia the cytokines IL-22, IL-20 and IL-24. IL-22R is capable of forming aheterodimeric complex with two different receptor partners, IL-10Rβ (orIL-10R2) and IL-20Rβ (or IL-20R2), and activation of these differentcomplexes by ligand binding triggers signalling via intracellulardownstream pathways.

FIG. 2 shows the full-length amino acid sequence of human IL-22R (SEQ IDNO: 71).

FIG. 3 shows the full-length nucleotide sequence encoding human IL-22R(SEQ ID NO: 72).

FIGS. 4A-4B show inhibition of IL-22 and IL-20 mediated signalling viaIL-22R in cell-based proliferation assays. In FIG. 4A, the effect ofIL-22R mAbs on proliferation of the BW-hIL-22R cell line is shown.BW-hIL-22R cells stably express the human IL-22R and growth isinhibited/arrested in response to the ligand IL-22. Antibodies capableof blocking the interaction between IL-22 and hIL-22R alleviate thegrowth inhibition mediated by ligand-receptor binding. In FIG. 4B, theeffect of IL-22R mAbs on proliferation of the Baf3-hIL-22R/IL20Rb cellline is shown. Baf3-hIL-22R/IL20Rb cells stably express the componentsof the receptor complex IL-22R/IL20Rb such that the cells proliferate inthe presence of IL-20. Antibodies capable of blocking the interactionbetween IL-20 and this receptor complex inhibit the proliferationinduced by ligand binding.

FIGS. 5A-5B show the alignment of the IL-22R extracellular domain fromvarious species. FIG. 5A shows the partial EST sequences available fromGenbank; FIG. 5B shows the sequences determined after cloning ofcynomolgus and rhesus IL-22R from the cynomolgus cDNA library.

FIG. 6 shows the results of the competitive ELISA experiments carriedout to map the epitopes of IL-22R mAbs. A variety of epitopes wereidentified for antibodies of VH families 1-8, 10, 11, 19 and 22. Theepitopes were grouped according to whether the antibodies (i) blockedIL-22 binding in vitro and neutralised IL-22 signalling in a cell-basedassay (bottom left quadrant); (ii) blocked IL-22 binding in vitro buthad no neutralising activity in the cell-based assay (top rightquandrant); or (iii) did not block IL-22 binding in vitro but did haveneutralising activity in a cell-based assay (bottom right quadrant).

FIGS. 7A-7B show the crystal structure of IL-22R in complex with IL-22.In FIG. 7A, domains D1 and D2 of IL-22R contribute residues to theinterface with the ligand IL-22. In FIG. 7B, Y60 in domain D1 is animportant IL-22R residue contributing to the interaction with site 1A ofthe IL-22 ligand.

FIG. 8 shows the inhibition of IL-20 mediated signalling via IL-22R in acell-based proliferation assay. Various IL-22R mAbs were tested fortheir ability to inhibit the IL-20 induced proliferation ofBaf3-hIL-22R/IL20Rb cells.

FIG. 9A-9B shows the inhibition of IL-22 and IL-20 mediated signallingvia IL-22R in cell-based assays. In FIG. 9A, the effect of IL-22R mAbson proliferation of the BW-hIL-22R cell line is shown. BW-hIL-22R cellsstably express the human IL-22R and growth is inhibited/arrested inresponse to the ligand IL-22. Antibodies capable of blocking theinteraction between IL-22 and hIL-22R alleviate the growth inhibitionmediated by ligand-receptor binding. In FIG. 9B, the effect of IL-22RmAbs on proliferation of the Baf3-hIL-22R/IL20Rb cell line is shown.Baf3-hIL-22R/IL20Rb cells stably express the components of the receptorcomplex IL-22R/IL20Rb such that the cells proliferate in the presence ofIL-20. Antibodies capable of blocking the interaction between IL-20 andthis receptor complex inhibit the proliferation induced by ligandbinding.

FIG. 10 shows schematically the results of the epitope mappingexperiments for the germlined IL-22R antibodies 230C9 and 223G5. NB“Zymo” is equivalent to 280-346-TSY″.

FIG. 11 shows cross-reactivity of the germlined antibodies for human andcynomolgus IL-22R, as determined by FACS analysis. Antibody 230C9cross-reacts with the human IL-22R and cyno IL-22R (left-hand panels)whereas antibody 223G5 binds to human IL-22R but does not cross-reactwith cyno IL-22R (right-hand panels).

FIG. 12 shows pharmacokinetic data for IL-22R antibody 230C9. Cynomolgusmonkeys were injected intravenously with a single 10 mg/kg dose ofantibody. Samples were taken at different time points and tested forplasma concentration of antibody by ELISA. Antibody 230C9 was found tohave a half-life of about 19.4 days.

FIGS. 13A-13B show pharmacokinetic data for IL-22R antibody 230C9 atvarious doses. At higher doses (≥10 mg/kg) when the capacity for targetmediated drug disposition (TMDD) is saturated, the clearance valuesapproach the non-specific clearance by RES. B. Clearance of 230C9-N297Qin cynomolgus. The total clearance represents the sum of 1) TMDD that isnon-linear and saturable and 2) non-specific clearance that is linearand attributed to RES. Plasma half-life has an inverse relationship withclearance, leading to a long half-live at high doses and shorterhalf-life at lower doses due to target mediated clearance.

FIGS. 14A-14B show the pharmacodynamic effect of the IL-22R antibody230C9. A cynomolgus monkey was exposed to 230C9 at different doses andthe effects on an IMQ-treated skin section and a normal skin section ofthe monkey were assessed. Increasing doses of antibody 230C9 were foundto normalize epidermal thickness (A) and reduce the frequency of Ki67positive nuclei (B) in the IMQ-treated skin section.

FIG. 15 shows the effect of the IL-22R antibody 230C9 (ARGX-112) onIL-22-regulated FLG2 mRNA levels in skin punch biopsies. Cynomolgusmonkeys were administered single IV infusions of antibody 230C9 atdifferent doses: 1 mg/kg, 5 mg/kg and 30 mg/kg (3 animals per dose).Recombinant human IL-22 reduced the total skin FLG2 mRNA levels whereasthis effect was reversed by antibody 230C9. The y axis indicatesrelative FLG2 expression as compared with a reference gene. Statisticalcomparisons between groups are indicated by the lines at the top of thegraph with confidence intervals as follows: *p<0.05; **p<0.01;***p<0.001. The percentages shown (60%, 64%, 60%, respectively) indicatethe % inhibition of the signal.

FIG. 16 shows the effect of the IL-22R antibody 230C9 (ARGX-112) onIL-22 regulated DEFB4 gene expression in human ex vivo skin explants.Abdominal skin explants were treated with increasing concentrations ofantibody 230C9 prior to stimulation with 20 ng/ml rhIL-22. Antibody230C9 was able to reverse the IL-22-mediated increase in DEFB4 mRNAlevels in a dose-dependent manner. The y axis indicates relative DEFB4expression as compared with a reference gene. LLOQ means lower level ofquantification.

FIGS. 17A-17B show the effect of the IL-22R antibody 230C9 (ARGX-112) onIL-22 regulated FLG2 and LOR gene expression in cynomolgus monkey exvivo skin explants. Skin biopsies were treated with increasingconcentrations of antibody 230C9 prior to stimulation with 20 ng/mlrhIL-22. Antibody 230C9 was able to reverse the IL-22-mediated decreasein FLG2 mRNA levels (A) and LOR mRNA levels (B) in a dose-dependentmanner. The y axes indicate relative FLG2 and LOR expression as comparedwith a reference gene.

FIG. 18 shows the effect of the IL-22R antibody 230C9 (ARGX-112) onprimary human keratinocytes. Keratinocytes were pre-treated with 230C9antibody and then stimulated with a mixture of IL-4, IL-13, IL-22 andIFN-γ. Control cells were treated with a mixture of IL-4, IL-13 andIFN-γ. The 230C9 antibody showed dose-dependent inhibition of CCL2secretion.

DETAILED DESCRIPTION A. Definitions

“Antibody” or “Immunoglobulin”—As used herein, the term “immunoglobulin”includes a polypeptide having a combination of two heavy and two lightchains whether or not it possesses any relevant specificimmunoreactivity. “Antibodies” refer to such assemblies which havesignificant known specific immunoreactive activity to an antigen ofinterest (e.g. the cytokine receptor IL-22R). The term “IL-22Rantibodies” is used herein to refer to antibodies which exhibitimmunological specificity for IL-22R protein, including human IL-22R andin some cases species homologues thereof. Antibodies and immunoglobulinscomprise light and heavy chains, with or without an interchain covalentlinkage between them. Basic immunoglobulin structures in vertebratesystems are relatively well understood.

The generic term “immunoglobulin” comprises five distinct classes ofantibody that can be distinguished biochemically. All five classes ofantibodies are within the scope of the present invention. The followingdiscussion will generally be directed to the IgG class of immunoglobulinmolecules. With regard to IgG, immunoglobulins comprise two identicallight polypeptide chains of molecular weight approximately 23,000Daltons, and two identical heavy chains of molecular weight53,000-70,000. The four chains are joined by disulfide bonds in a “Y”configuration wherein the light chains bracket the heavy chains startingat the mouth of the “Y” and continuing through the variable region.

The light chains of an antibody are classified as either kappa or lambda(κ,λ). Each heavy chain class may be bound with either a kappa or lambdalight chain. In general, the light and heavy chains are covalentlybonded to each other, and the “tail” portions of the two heavy chainsare bonded to each other by covalent disulfide linkages or non-covalentlinkages when the immunoglobulins are generated either by hybridomas, Bcells or genetically engineered host cells. In the heavy chain, theamino acid sequences run from an N-terminus at the forked ends of the Yconfiguration to the C-terminus at the bottom of each chain. Thoseskilled in the art will appreciate that heavy chains are classified asgamma, mu, alpha, delta, or epsilon, (γ, μ, α, δ, ε) with somesubclasses among them (e.g., γ1-γ4). It is the nature of this chain thatdetermines the “class” of the antibody as IgG, IgM, IgA, IgD or IgE,respectively. The immunoglobulin subclasses (isotypes) e.g., IgG1, IgG2,IgG3, IgG4, IgA1, etc. are well characterized and are known to conferfunctional specialization. Modified versions of each of these classesand isotypes are readily discernible to the skilled artisan in view ofthe instant disclosure and, accordingly, are within the scope of theinstant invention.

As indicated above, the variable region of an antibody allows theantibody to selectively recognize and specifically bind epitopes onantigens. That is, the VL domain and VH domain of an antibody combine toform the variable region that defines a three dimensional antigenbinding site. This quaternary antibody structure forms the antigenbinding site present at the end of each arm of the Y. More specifically,the antigen binding site is defined by three complementary determiningregions (CDRs) on each of the VH and VL chains.

“IL-22R”—As used herein, the term “IL-22R” means the type II cytokinereceptor that mediates signalling via the ligands IL-22, IL-20 andIL-24. IL-22R is capable of forming heterodimeric complexes at the cellsurface with IL-10R2 and IL-20R2. IL-22R may also be referred to asIL22R, IL-22R1, IL22R1, IL22RA, IL-22RA, CRF2-9 and Zcytor 11. The term“IL-22R1” is broad enough to cover the human form of the receptor andspecies homologues. The amino acid sequence of the full-length humanIL-22R is represented by SEQ ID NO: 71 and the encoding nucleotidesequence is represented by SEQ ID NO: 72 (see FIGS. 2 and 3). Thesesequences correspond to the sequences deposited in the SwissProtdatabase as human protein Interleukin-22 receptor subunit, accessionnumber Q8N6P7.

“Binding Site”—As used herein, the term “binding site” comprises aregion of a polypeptide which is responsible for selectively binding toa target antigen of interest (e.g. IL-22R). Binding domains comprise atleast one binding site. Exemplary binding domains include an antibodyvariable domain. The antibody molecules of the invention may comprise asingle binding site or multiple (e.g., two, three or four) bindingsites.

“Derived From”—As used herein the term “derived from” a designatedprotein (e.g. a camelid antibody or antigen-binding fragment thereof)refers to the origin of the polypeptide or amino acid sequence. In oneembodiment, the polypeptide or amino acid sequence which is derived froma particular starting polypeptide is a CDR sequence or sequence relatedthereto. In one embodiment, the amino acid sequence which is derivedfrom a particular starting polypeptide is not contiguous. For example,in one embodiment, one, two, three, four, five, or six CDRs are derivedfrom a starting antibody. In one embodiment, the polypeptide or aminoacid sequence which is derived from a particular starting polypeptide oramino acid sequence has an amino acid sequence that is essentiallyidentical to that of the starting sequence, or a portion thereof whereinthe portion consists of at least 3-5 amino acids, at least 5-10 aminoacids, at least 10-20 amino acids, at least 20-30 amino acids, or atleast 30-50 amino acids, or which is otherwise identifiable to one ofordinary skill in the art as having its origin in the starting sequence.In one embodiment, the one or more CDR sequences derived from thestarting antibody are altered to produce variant CDR sequences, e.g.affinity variants, wherein the variant CDR sequences maintain targetantigen binding activity.

“Camelid-Derived”—In certain preferred embodiments, the antibodies ofthe invention comprise framework amino acid sequences and/or CDR aminoacid sequences derived from a camelid conventional antibody raised byactive immunisation of a camelid. However, antibodies of the inventioncomprising camelid-derived amino acid sequences may be engineered tocomprise framework and/or constant region sequences derived from a humanamino acid sequence (i.e. a human antibody) or other non-camelidmammalian species. For example, a human or non-human primate frameworkregion, heavy chain portion, and/or hinge portion may be included in thesubject IL-22R antibodies. In one embodiment, one or more non-camelidamino acids may be present in the framework region of a“camelid-derived” antibody, e.g., a camelid framework amino acidsequence may comprise one or more amino acid mutations in which thecorresponding human or non-human primate amino acid residue is present.Moreover, camelid-derived VH and VL domains, or humanised variantsthereof, may be linked to the constant domains of human antibodies toproduce a chimeric molecule, as described elsewhere herein.

“Conservative amino acid substitution”—A “conservative amino acidsubstitution” is one in which the amino acid residue is replaced with anamino acid residue having a similar side chain. Families of amino acidresidues having similar side chains have been defined in the art,including basic side chains (e.g., lysine, arginine, histidine), acidicside chains (e.g., aspartic acid, glutamic acid), uncharged polar sidechains (e.g., glycine, asparagine, glutamine, serine, threonine,tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). Thus, a nonessential amino acid residue in an immunoglobulinpolypeptide may be replaced with another amino acid residue from thesame side chain family. In another embodiment, a string of amino acidscan be replaced with a structurally similar string that differs in orderand/or composition of side chain family members.

“Heavy chain portion”—As used herein, the term “heavy chain portion”includes amino acid sequences derived from the constant domains of animmunoglobulin heavy chain. A polypeptide comprising a heavy chainportion comprises at least one of: a CH1 domain, a hinge (e.g., upper,middle, and/or lower hinge region) domain, a CH2 domain, a CH3 domain,or a variant or fragment thereof. In one embodiment, an antibody orantigen binding fragment of the invention may comprise the Fc portion ofan immunoglobulin heavy chain (e.g., a hinge portion, a CH2 domain, anda CH3 domain). In another embodiment, an antibody or antigen bindingfragment of the invention may lack at least a portion of a constantdomain (e.g., all or part of a CH2 domain). In certain embodiments, atleast one, and preferably all, of the constant domains are derived froma human immunoglobulin heavy chain. For example, in one preferredembodiment, the heavy chain portion comprises a fully human hingedomain. In other preferred embodiments, the heavy chain portioncomprising a fully human Fc portion (e.g., hinge, CH2 and CH3 domainsequences from a human immunoglobulin).

In certain embodiments, the constituent constant domains of the heavychain portion are from different immunoglobulin molecules. For example,a heavy chain portion of a polypeptide may comprise a CH2 domain derivedfrom an IgG1 molecule and a hinge region derived from an IgG3 or IgG4molecule. In other embodiments, the constant domains are chimericdomains comprising portions of different immunoglobulin molecules. Forexample, a hinge may comprise a first portion from an IgG1 molecule anda second portion from an IgG3 or IgG4 molecule. As set forth above, itwill be understood by one of ordinary skill in the art that the constantdomains of the heavy chain portion may be modified such that they varyin amino acid sequence from the naturally occurring (wild-type)immunoglobulin molecule. That is, the polypeptides of the inventiondisclosed herein may comprise alterations or modifications to one ormore of the heavy chain constant domains (CH1, hinge, CH2 or CH3) and/orto the light chain constant region domain (CL). Exemplary modificationsinclude additions, deletions or substitutions of one or more amino acidsin one or more domains.

“Chimeric”—A “chimeric” protein comprises a first amino acid sequencelinked to a second amino acid sequence with which it is not naturallylinked in nature. The amino acid sequences may normally exist inseparate proteins that are brought together in the fusion polypeptide orthey may normally exist in the same protein but are placed in a newarrangement in the fusion polypeptide. A chimeric protein may becreated, for example, by chemical synthesis, or by creating andtranslating a polynucleotide in which the peptide regions are encoded inthe desired relationship. Exemplary chimeric antibodies of the inventioninclude fusion proteins comprising camelid-derived VH and VL domains, orhumanised variants thereof, fused to the constant domains of a humanantibody, e.g. human IgG1, IgG2, IgG3 or IgG4.

“Variable region” or “variable domain”—The terms “variable region” and“variable domain” are used herein interchangeably and are intended tohave equivalent meaning. The term “variable” refers to the fact thatcertain portions of the variable domains VH and VL differ extensively insequence among antibodies and are used in the binding and specificity ofeach particular antibody for its target antigen. However, thevariability is not evenly distributed throughout the variable domains ofantibodies. It is concentrated in three segments called “hypervariableloops” in each of the VL domain and the VH domain which form part of theantigen binding site. The first, second and third hypervariable loops ofthe VLambda light chain domain are referred to herein as L1(λ), L2(λ)and L3(λ) and may be defined as comprising residues 24-33 (L1(λ),consisting of 9, 10 or 11 amino acid residues), 49-53 (L2(λ), consistingof 3 residues) and 90-96 (L3(λ), consisting of 5 residues) in the VLdomain (Morea et al., Methods 20:267-279 (2000)). The first, second andthird hypervariable loops of the VKappa light chain domain are referredto herein as L1(κ), L2(κ) and L3(κ) and may be defined as comprisingresidues 25-33 (L1(κ), consisting of 6, 7, 8, 11, 12 or 13 residues),49-53 (L2(κ), consisting of 3 residues) and 90-97 (L3(κ), consisting of6 residues) in the VL domain (Morea et al., Methods 20:267-279 (2000)).The first, second and third hypervariable loops of the VH domain arereferred to herein as H1, H2 and H3 and may be defined as comprisingresidues 25-33 (H1, consisting of 7, 8 or 9 residues), 52-56 (H2,consisting of 3 or 4 residues) and 91-105 (H3, highly variable inlength) in the VH domain (Morea et al., Methods 20:267-279 (2000)).

Unless otherwise indicated, the terms L1, L2 and L3 respectively referto the first, second and third hypervariable loops of a VL domain, andencompass hypervariable loops obtained from both Vkappa and Vlambdaisotypes. The terms H1, H2 and H3 respectively refer to the first,second and third hypervariable loops of the VH domain, and encompasshypervariable loops obtained from any of the known heavy chain isotypes,including γ, ε, δ, α or μ.

The hypervariable loops L1, L2, L3, H1, H2 and H3 may each comprise partof a “complementarity determining region” or “CDR”, as defined below.The terms “hypervariable loop” and “complementarity determining region”are not strictly synonymous, since the hypervariable loops (HVs) aredefined on the basis of structure, whereas complementarity determiningregions (CDRs) are defined based on sequence variability (Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md., 1983) and thelimits of the HVs and the CDRs may be different in some VH and VLdomains.

The CDRs of the VL and VH domains can typically be defined as comprisingthe following amino acids: residues 24-34 (LCDR1), 50-56 (LCDR2) and89-97 (LCDR3) in the light chain variable domain, and residues 31-35 or31-35b (HCDR1), 50-65 (HCDR2) and 95-102 (HCDR3) in the heavy chainvariable domain; (Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, National Institutes of Health,Bethesda, Md. (1991)). Thus, the HVs may be comprised within thecorresponding CDRs and references herein to the “hypervariable loops” ofVH and VL domains should be interpreted as also encompassing thecorresponding CDRs, and vice versa, unless otherwise indicated.

The more highly conserved portions of variable domains are called theframework region (FR), as defined below. The variable domains of nativeheavy and light chains each comprise four FRs (FR1, FR2, FR3 and FR4,respectively), largely adopting a β-sheet configuration, connected bythe three hypervariable loops. The hypervariable loops in each chain areheld together in close proximity by the FRs and, with the hypervariableloops from the other chain, contribute to the formation of theantigen-binding site of antibodies. Structural analysis of antibodiesrevealed the relationship between the sequence and the shape of thebinding site formed by the complementarity determining regions (Chothiaet al., J. Mol. Biol. 227: 799-817 (1992)); Tramontano et al., J. Mol.Biol, 215:175-182 (1990)). Despite their high sequence variability, fiveof the six loops adopt just a small repertoire of main-chainconformations, called “canonical structures”. These conformations arefirst of all determined by the length of the loops and secondly by thepresence of key residues at certain positions in the loops and in theframework regions that determine the conformation through their packing,hydrogen bonding or the ability to assume unusual main-chainconformations.

“CDR”—As used herein, the term “CDR” or “complementarity determiningregion” means the non-contiguous antigen combining sites found withinthe variable region of both heavy and light chain polypeptides. Theseparticular regions have been described by Kabat et al., J. Biol. Chem.252, 6609-6616 (1977) and Kabat et al., Sequences of protein ofimmunological interest. (1991), and by Chothia et al., J. Mol. Biol.196:901-917 (1987) and by MacCallum et al., J. Mol. Biol. 262:732-745(1996) where the definitions include overlapping or subsets of aminoacid residues when compared against each other. The amino acid residueswhich encompass the CDRs as defined by each of the above citedreferences are set forth for comparison. Preferably, the term “CDR” is aCDR as defined by Kabat based on sequence comparisons.

TABLE 1 CDR definitions CDR Definitions Kabat¹ Chothia² MacCallum³ V_(H)CDR1 31-35 26-32 30-35 V_(H) CDR2 50-65 53-55 47-58 V_(H) CDR3  95-102 96-101  93-101 V_(L) CDR1 24-34 26-32 30-36 V_(L) CDR2 50-56 50-5246-55 V_(L) CDR3 89-97 91-96 89-96 ¹Residue numbering follows thenomenclature of Kabat et al., supra ²Residue numbering follows thenomenclature of Chothia et al., supra ³Residue numbering follows thenomenclature of MacCallum et al., supra

“Framework region”—The term “framework region” or “FR region” as usedherein, includes the amino acid residues that are part of the variableregion, but are not part of the CDRs (e.g., using the Kabat definitionof CDRs). Therefore, a variable region framework is between about100-120 amino acids in length but includes only those amino acidsoutside of the CDRs. For the specific example of a heavy chain variabledomain and for the CDRs as defined by Kabat et al., framework region 1corresponds to the domain of the variable region encompassing aminoacids 1-30; framework region 2 corresponds to the domain of the variableregion encompassing amino acids 36-49; framework region 3 corresponds tothe domain of the variable region encompassing amino acids 66-94, andframework region 4 corresponds to the domain of the variable region fromamino acids 103 to the end of the variable region. The framework regionsfor the light chain are similarly separated by each of the light chainvariable region CDRs. Similarly, using the definition of CDRs by Chothiaet al. or McCallum et al. the framework region boundaries are separatedby the respective CDR termini as described above. In preferredembodiments the CDRs are as defined by Kabat.

In naturally occurring antibodies, the six CDRs present on eachmonomeric antibody are short, non-contiguous sequences of amino acidsthat are specifically positioned to form the antigen binding site as theantibody assumes its three dimensional configuration in an aqueousenvironment. The remainder of the heavy and light variable domains showless inter-molecular variability in amino acid sequence and are termedthe framework regions. The framework regions largely adopt a β-sheetconformation and the CDRs form loops which connect, and in some casesform part of, the β-sheet structure. Thus, these framework regions actto form a scaffold that provides for positioning the six CDRs in correctorientation by inter-chain, non-covalent interactions. The antigenbinding site formed by the positioned CDRs defines a surfacecomplementary to the epitope on the immunoreactive antigen. Thiscomplementary surface promotes the non-covalent binding of the antibodyto the immunoreactive antigen epitope. The position of CDRs can bereadily identified by one of ordinary skill in the art.

“Hinge region”—As used herein, the term “hinge region” includes theportion of a heavy chain molecule that joins the CH1 domain to the CH2domain. This hinge region comprises approximately 25 residues and isflexible, thus allowing the two N-terminal antigen binding regions tomove independently. Hinge regions can be subdivided into three distinctdomains: upper, middle, and lower hinge domains (Roux K. H. et al. J.Immunol. 161:4083-90 1998). Antibodies of the invention comprising a“fully human” hinge region may contain one of the hinge region sequencesshown in Table 2 below.

TABLE 2 Human hinge sequences Upper Middle Lower IgG hinge hinge hingeIgG1 EPKSCDKTHT CPPCP APELLGGP (SEQ ID (SEQ ID (SEQ ID NO: 82) NO: 83)NO: 84) IgG3 ELKTPL CPRCP APELLGGP GDTTHT (EPKSCDTPPPCPRCP)₃ (SEQ ID(SEQ ID (SEQ ID NO: 87) NO: 85) NO: 86) IgG4 ESKYGPP CPSCP APEFLGGP(SEQ ID (SEQ ID (SEQ ID NO: 88) NO: 89) NO: 90) IgG42 ERK CCVECPPPCPAPPVAGP (SEQ ID (SEQ ID (SEQ ID NO: 91) NO: 92) NO: 93)

“CH2 domain”—As used herein the term “CH2 domain” includes the portionof a heavy chain molecule that extends, e.g., from about residue 244 toresidue 360 of an antibody using conventional numbering schemes(residues 244 to 360, Kabat numbering system; and residues 231-340, EUnumbering system, Kabat E A et al. Sequences of Proteins ofImmunological Interest. Bethesda, US Department of Health and HumanServices, NIH. 1991). The CH2 domain is unique in that it is not closelypaired with another domain. Rather, two N-linked branched carbohydratechains are interposed between the two CH2 domains of an intact nativeIgG molecule. It is also well documented that the CH3 domain extendsfrom the CH2 domain to the C-terminal of the IgG molecule and comprisesapproximately 108 residues.

“Fragment”—The term “fragment”, as used in the context of antibodies ofthe invention, refers to a part or portion of an antibody or antibodychain comprising fewer amino acid residues than an intact or completeantibody or antibody chain. The term “antigen-binding fragment” refersto a polypeptide fragment of an immunoglobulin or antibody that bindsantigen or competes with intact antibody (i.e., with the intact antibodyfrom which they were derived) for antigen binding (i.e., specificbinding to IL-22R). As used herein, the term “fragment” of an antibodymolecule includes antigen-binding fragments of antibodies, for example,an antibody light chain variable domain (VL), an antibody heavy chainvariable domain (VH), a single chain antibody (scFv), a F(ab′)2fragment, a Fab fragment, an Fd fragment, an Fv fragment, and a singledomain antibody fragment (DAb). Fragments can be obtained, e.g., viachemical or enzymatic treatment of an intact or complete antibody orantibody chain or by recombinant means.

“Valency”—As used herein the term “valency” refers to the number ofpotential target binding sites in a polypeptide. Each target bindingsite specifically binds one target molecule or specific site on a targetmolecule. When a polypeptide comprises more than one target bindingsite, each target binding site may specifically bind the same ordifferent molecules (e.g., may bind to different ligands or differentantigens, or different epitopes on the same antigen).

“Specificity”—The term “specificity” refers to the ability to bind(e.g., immunoreact with) a given target, e.g., IL-22R. A polypeptide maybe monospecific and contain one or more binding sites which specificallybind a target or a polypeptide may be multispecific and contain two ormore binding sites which specifically bind the same or differenttargets.

“Synthetic”—As used herein the term “synthetic” with respect topolypeptides includes polypeptides which comprise an amino acid sequencethat is not naturally occurring. For example, non-naturally occurringpolypeptides which are modified forms of naturally occurringpolypeptides (e.g., comprising a mutation such as an addition,substitution or deletion) or which comprise a first amino acid sequence(which may or may not be naturally occurring) that is linked in a linearsequence of amino acids to a second amino acid sequence (which may ormay not be naturally occurring) to which it is not naturally linked innature.

“Engineered”—As used herein the term “engineered” includes manipulationof nucleic acid or polypeptide molecules by synthetic means (e.g. byrecombinant techniques, in vitro peptide synthesis, by enzymatic orchemical coupling of peptides or some combination of these techniques).Preferably, the antibodies of the invention are engineered, includingfor example, humanized and/or chimeric antibodies, and antibodies whichhave been engineered to improve one or more properties, such as antigenbinding, stability/half-life or effector function.

“Modified antibody”—As used herein, the term “modified antibody”includes synthetic forms of antibodies which are altered such that theyare not naturally occurring, e.g., antibodies that comprise at least twoheavy chain portions but not two complete heavy chains (such as, domaindeleted antibodies or minibodies); multispecific forms of antibodies(e.g., bispecific, trispecific, etc.) altered to bind to two or moredifferent antigens or to different epitopes on a single antigen); heavychain molecules joined to scFv molecules and the like. scFv moleculesare known in the art and are described, e.g., in U.S. Pat. No.5,892,019. In addition, the term “modified antibody” includesmultivalent forms of antibodies (e.g., trivalent, tetravalent, etc.,antibodies that bind to three or more copies of the same antigen). Inanother embodiment, a modified antibody of the invention is a fusionprotein comprising at least one heavy chain portion lacking a CH2 domainand comprising a binding domain of a polypeptide comprising the bindingportion of one member of a receptor ligand pair.

The term “modified antibody” may also be used herein to refer to aminoacid sequence variants of the antibodies of the invention asstructurally defined herein. It will be understood by one of ordinaryskill in the art that an antibody may be modified to produce a variantantibody which varies in amino acid sequence in comparison to theantibody from which it was derived. For example, nucleotide or aminoacid substitutions leading to conservative substitutions or changes at“non-essential” amino acid residues may be made (e.g., in CDR and/orframework residues). Amino acid substitutions can include replacement ofone or more amino acids with a naturally occurring or non-natural aminoacid.

“Humanising substitutions”—As used herein, the term “humanisingsubstitutions” refers to amino acid substitutions in which the aminoacid residue present at a particular position in the VH or VL domain ofan antibody (for example a camelid-derived IL-22R antibody) is replacedwith an amino acid residue which occurs at an equivalent position in areference human VH or VL domain. The reference human VH or VL domain maybe a VH or VL domain encoded by the human germline. Humanisingsubstitutions may be made in the framework regions and/or the CDRs ofthe antibodies, defined herein.

“Humanised variants”—As used herein the term “humanised variant” refersto a variant antibody which contains one or more “humanisingsubstitutions” compared to a reference antibody, wherein a portion ofthe reference antibody (e.g. the VH domain and/or the VL domain or partsthereof containing at least one CDR) has an amino acid derived from anon-human species, and the “humanising substitutions” occur within theamino acid sequence derived from a non-human species.

“Germlined variants”—The term “germlined variant” is used herein torefer specifically to “humanised variants” in which the “humanisingsubstitutions” result in replacement of one or more amino acid residuespresent at a particular position (s) in the VH or VL domain of anantibody (for example a camelid-derived IL-22R antibody) with an aminoacid residue which occurs at an equivalent position in a reference humanVH or VL domain encoded by the human germline. It is typical that forany given “germlined variant”, the replacement amino acid residuessubstituted into the germlined variant are taken exclusively, orpredominantly, from a single human germline-encoded VH or VL domain. Theterms “humanised variant” and “germlined variant” are often usedinterchangeably herein. Introduction of one or more “humanisingsubstitutions” into a camelid-derived (e.g. llama derived) VH or VLdomain results in production of a “humanised variant” of the camelid(llama)-derived VH or VL domain. If the amino acid residues substitutedin are derived predominantly or exclusively from a single humangermline-encoded VH or VL domain sequence, then the result may be a“human germlined variant” of the camelid (llama)-derived VH or VLdomain.

“Affinity variants”—As used herein, the term “affinity variant” refersto a variant antibody which exhibits one or more changes in amino acidsequence compared to a reference antibody, wherein the affinity variantexhibits an altered affinity for the target antigen in comparison to thereference antibody. For example, affinity variants will exhibit achanged affinity for IL-22R, as compared to the reference IL-22Rantibody. Preferably the affinity variant will exhibit improved affinityfor the target antigen, e.g. IL-22R, as compared to the referenceantibody. Affinity variants typically exhibit one or more changes inamino acid sequence in the CDRs, as compared to the reference antibody.Such substitutions may result in replacement of the original amino acidpresent at a given position in the CDRs with a different amino acidresidue, which may be a naturally occurring amino acid residue or anon-naturally occurring amino acid residue. The amino acid substitutionsmay be conservative or non-conservative.

“High human homology”—An antibody comprising a heavy chain variabledomain (VH) and a light chain variable domain (VL) will be considered ashaving high human homology if the VH domains and the VL domains, takentogether, exhibit at least 90% amino acid sequence identity to theclosest matching human germline VH and VL sequences. Antibodies havinghigh human homology may include antibodies comprising VH and VL domainsof native non-human antibodies which exhibit sufficiently high %sequence identity to human germline sequences, including for exampleantibodies comprising VH and VL domains of camelid conventionalantibodies, as well as engineered, especially humanised or germlined,variants of such antibodies and also “fully human” antibodies.

In one embodiment the VH domain of the antibody with high human homologymay exhibit an amino acid sequence identity or sequence homology of 80%or greater with one or more human VH domains across the frameworkregions FR1, FR2, FR3 and FR4. In other embodiments the amino acidsequence identity or sequence homology between the VH domain of thepolypeptide of the invention and the closest matching human germline VHdomain sequence may be 85% or greater, 90% or greater, 95% or greater,97% or greater, or up to 99% or even 100%.

In one embodiment the VH domain of the antibody with high human homologymay contain one or more (e.g. 1 to 10) amino acid sequence mis-matchesacross the framework regions FR1, FR2, FR3 and FR4, in comparison to theclosest matched human VH sequence.

In another embodiment the VL domain of the antibody with high humanhomology may exhibit a sequence identity or sequence homology of 80% orgreater with one or more human VL domains across the framework regionsFR1, FR2, FR3 and FR4. In other embodiments the amino acid sequenceidentity or sequence homology between the VL domain of the polypeptideof the invention and the closest matching human germline VL domainsequence may be 85% or greater 90% or greater, 95% or greater, 97% orgreater, or up to 99% or even 100%.

In one embodiment the VL domain of the antibody with high human homologymay contain one or more (e.g. 1 to 10) amino acid sequence mis-matchesacross the framework regions FR1, FR2, FR3 and FR4, in comparison to theclosest matched human VL sequence.

Before analyzing the percentage sequence identity between the antibodywith high human homology and human germline VH and VL, the canonicalfolds may be determined, which allows the identification of the familyof human germline segments with the identical combination of canonicalfolds for H1 and H2 or L1 and L2 (and L3). Subsequently the humangermline family member that has the highest degree of sequence homologywith the variable region of the antibody of interest is chosen forscoring the sequence homology. The determination of Chothia canonicalclasses of hypervariable loops L1, L2, L3, H1 and H2 can be performedwith the bioinformatics tools publicly available on webpagewww.bioinf.org.uk/abs/chothia.html.page. The output of the program showsthe key residue requirements in a datafile. In these datafiles, the keyresidue positions are shown with the allowed amino acids at eachposition. The sequence of the variable region of the antibody ofinterest is given as input and is first aligned with a consensusantibody sequence to assign the Kabat numbering scheme. The analysis ofthe canonical folds uses a set of key residue templates derived by anautomated method developed by Martin and Thornton (Martin et al., J.Mol. Biol. 263:800-815 (1996)).

With the particular human germline V segment known, which uses the samecombination of canonical folds for H1 and H2 or L1 and L2 (and L3), thebest matching family member in terms of sequence homology can bedetermined. With bioinformatics tools the percentage sequence identitybetween the VH and VL domain framework amino acid sequences of theantibody of interest and corresponding sequences encoded by the humangermline can be determined, but actually manual alignment of thesequences can be applied as well. Human immunoglobulin sequences can beidentified from several protein data bases, such as VBase(http://vbase.mrc-cpe.cam.ac.uk/) or the Pluckthun/Honegger database(http://www.bioc.unizh.ch/antibody/Sequences/Germlines. To compare thehuman sequences to the V regions of VH or VL domains in an antibody ofinterest a sequence alignment algorithm such as available via websiteslike www.expasy.ch/tools/#align can be used, but also manual alignmentwith the limited set of sequences can be performed. Human germline lightand heavy chain sequences of the families with the same combinations ofcanonical folds and with the highest degree of homology with theframework regions 1, 2, and 3 of each chain are selected and comparedwith the variable region of interest; also the FR4 is checked againstthe human germline JH and JK or JL regions.

Note that in the calculation of overall percent sequence homology theresidues of FR1, FR2 and FR3 are evaluated using the closest matchsequence from the human germline family with the identical combinationof canonical folds. Only residues different from the closest match orother members of the same family with the same combination of canonicalfolds are scored (NB—excluding any primer-encoded differences). However,for the purposes of humanization, residues in framework regionsidentical to members of other human germline families, which do not havethe same combination of canonical folds, can be considered “human”,despite the fact that these are scored “negative” according to thestringent conditions described above. This assumption is based on the“mix and match” approach for humanization, in which each of FR1, FR2,FR3 and FR4 is separately compared to its closest matching humangermline sequence and the humanized molecule therefore contains acombination of different FRs as was done by Qu and colleagues (Qu etla., Clin. Cancer Res. 5:3095-3100 (1999)) and Ono and colleagues (Onoet al., Mol. Immunol. 36:387-395 (1999)). The boundaries of theindividual framework regions may be assigned using the IMGT numberingscheme, which is an adaptation of the numbering scheme of Chothia(Lefranc et al., NAR 27: 209-212 (1999); imgt.cines.fr).

Antibodies with high human homology may comprise hypervariable loops orCDRs having human or human-like canonical folds, as discussed in detailbelow.

In one embodiment at least one hypervariable loop or CDR in either theVH domain or the VL domain of the antibody with high human homology maybe obtained or derived from a VH or VL domain of a non-human antibody,for example a conventional antibody from a species of Camelidae, yetexhibit a predicted or actual canonical fold structure which issubstantially identical to a canonical fold structure which occurs inhuman antibodies.

It is well established in the art that although the primary amino acidsequences of hypervariable loops present in both VH domains and VLdomains encoded by the human germline are, by definition, highlyvariable, all hypervariable loops, except CDR H3 of the VH domain, adoptonly a few distinct structural conformations, termed canonical folds(Chothia et al., J. Mol. Biol. 196:901-917 (1987); Tramontano et al.Proteins 6:382-94 (1989)), which depend on both the length of thehypervariable loop and presence of the so-called canonical amino acidresidues (Chothia et al., J. Mol. Biol. 196:901-917 (1987)). Actualcanonical structures of the hypervariable loops in intact VH or VLdomains can be determined by structural analysis (e.g. X-raycrystallography), but it is also possible to predict canonical structureon the basis of key amino acid residues which are characteristic of aparticular structure (discussed further below). In essence, the specificpattern of residues that determines each canonical structure forms a“signature” which enables the canonical structure to be recognised inhypervariable loops of a VH or VL domain of unknown structure; canonicalstructures can therefore be predicted on the basis of primary amino acidsequence alone.

The predicted canonical fold structures for the hypervariable loops ofany given VH or VL sequence in an antibody with high human homology canbe analysed using algorithms which are publicly available fromwww.bioinf.org.uk/abs/chothia.html,www.biochem.ucl.ac.uk/˜martin/antibodies.html andwww.bioc.unizh.ch/antibody/Sequences/Germlines/Vbase_hVk.html. Thesetools permit query VH or VL sequences to be aligned against human VH orVL domain sequences of known canonical structure, and a prediction ofcanonical structure made for the hypervariable loops of the querysequence.

In the case of the VH domain, H1 and H2 loops may be scored as having acanonical fold structure “substantially identical” to a canonical foldstructure known to occur in human antibodies if at least the first, andpreferably both, of the following criteria are fulfilled:

1. An identical length, determined by the number of residues, to theclosest matching human canonical structural class.

2. At least 33% identity, preferably at least 50% identity with the keyamino acid residues described for the corresponding human H1 and H2canonical structural classes.

(note for the purposes of the foregoing analysis the H1 and H2 loops aretreated separately and each compared against its closest matching humancanonical structural class).

The foregoing analysis relies on prediction of the canonical structureof the H1 and H2 loops of the antibody of interest. If the actualstructures of the H1 and H2 loops in the antibody of interest are known,for example based on X-ray crystallography, then the H1 and H2 loops inthe antibody of interest may also be scored as having a canonical foldstructure “substantially identical” to a canonical fold structure knownto occur in human antibodies if the length of the loop differs from thatof the closest matching human canonical structural class (typically by±1 or ±2 amino acids) but the actual structure of the H1 and H2 loops inthe antibody of interest matches the structure of a human canonicalfold.

Key amino acid residues found in the human canonical structural classesfor the first and second hypervariable loops of human VH domains (H1 andH2) are described by Chothia et al., J. Mol. Biol. 227:799-817 (1992),the contents of which are incorporated herein in their entirety byreference. In particular, Table 3 on page 802 of Chothia et al., whichis specifically incorporated herein by reference, lists preferred aminoacid residues at key sites for H1 canonical structures found in thehuman germline, whereas Table 4 on page 803, also specificallyincorporated by reference, lists preferred amino acid residues at keysites for CDR H2 canonical structures found in the human germline.

In one embodiment, both H1 and H2 in the VH domain of the antibody withhigh human homology exhibit a predicted or actual canonical foldstructure which is substantially identical to a canonical fold structurewhich occurs in human antibodies.

Antibodies with high human homology may comprise a VH domain in whichthe hypervariable loops H1 and H2 form a combination of canonical foldstructures which is identical to a combination of canonical structuresknown to occur in at least one human germline VH domain. It has beenobserved that only certain combinations of canonical fold structures atH1 and H2 actually occur in VH domains encoded by the human germline. Inan embodiment H1 and H2 in the VH domain of the antibody with high humanhomology may be obtained from a VH domain of a non-human species, e.g. aCamelidae species, yet form a combination of predicted or actualcanonical fold structures which is identical to a combination ofcanonical fold structures known to occur in a human germline orsomatically mutated VH domain. In non-limiting embodiments H1 and H2 inthe VH domain of the antibody with high human homology may be obtainedfrom a VH domain of a non-human species, e.g. a Camelidae species, andform one of the following canonical fold combinations: 1-1, 1-2, 1-3,1-6, 1-4, 2-1, 3-1 and 3-5.

An antibody with high human homology may contain a VH domain whichexhibits both high sequence identity/sequence homology with human VH,and which contains hypervariable loops exhibiting structural homologywith human VH.

It may be advantageous for the canonical folds present at H1 and H2 inthe VH domain of the antibody with high human homology, and thecombination thereof, to be “correct” for the human VH germline sequencewhich represents the closest match with the VH domain of the antibodywith high human homology in terms of overall primary amino acid sequenceidentity. By way of example, if the closest sequence match is with ahuman germline VH3 domain, then it may be advantageous for H1 and H2 toform a combination of canonical folds which also occurs naturally in ahuman VH3 domain. This may be particularly important in the case ofantibodies with high human homology which are derived from non-humanspecies, e.g. antibodies containing VH and VL domains which are derivedfrom camelid conventional antibodies, especially antibodies containinghumanised camelid VH and VL domains.

Thus, in one embodiment the VH domain of an IL-22R antibody with highhuman homology may exhibit a sequence identity or sequence homology of80% or greater, 85% or greater, 90% or greater, 95% or greater, 97% orgreater, or up to 99% or even 100% with a human VH domain across theframework regions FR1, FR2, FR3 and FR4, and in addition H1 and H2 inthe same antibody are obtained from a non-human VH domain (e.g. derivedfrom a Camelidae species), but form a combination of predicted or actualcanonical fold structures which is the same as a canonical foldcombination known to occur naturally in the same human VH domain.

In other embodiments, L1 and L2 in the VL domain of the antibody withhigh human homology are each obtained from a VL domain of a non-humanspecies (e.g. a camelid-derived VL domain), and each exhibits apredicted or actual canonical fold structure which is substantiallyidentical to a canonical fold structure which occurs in humanantibodies.

As with the VH domains, the hypervariable loops of VL domains of bothVLambda and VKappa types can adopt a limited number of conformations orcanonical structures, determined in part by length and also by thepresence of key amino acid residues at certain canonical positions.

Within an antibody of interest having high human homology, L1, L2 and L3loops obtained from a VL domain of a non-human species, e.g. a Camelidaespecies, may be scored as having a canonical fold structure“substantially identical” to a canonical fold structure known to occurin human antibodies if at least the first, and preferable both, of thefollowing criteria are fulfilled:

1. An identical length, determined by the number of residues, to theclosest matching human structural class.

2. At least 33% identity, preferably at least 50% identity with the keyamino acid residues described for the corresponding human L1 or L2canonical structural classes, from either the VLambda or the VKapparepertoire.

(Note for the purposes of the foregoing analysis the L1 and L2 loops aretreated separately and each compared against its closest matching humancanonical structural class.)

The foregoing analysis relies on prediction of the canonical structureof the L1, L2 and L3 loops in the VL domain of the antibody of interest.If the actual structure of the L1, L2 and L3 loops is known, for examplebased on X-ray crystallography, then L1, L2 or L3 loops derived from theantibody of interest may also be scored as having a canonical foldstructure “substantially identical” to a canonical fold structure knownto occur in human antibodies if the length of the loop differs from thatof the closest matching human canonical structural class (typically by±1 or ±2 amino acids) but the actual structure of the Camelidae loopsmatches a human canonical fold.

Key amino acid residues found in the human canonical structural classesfor the CDRs of human VLambda and VKappa domains are described by Moreaet al. Methods, 20: 267-279 (2000) and Martin et al., J. Mol. Biol.,263:800-815 (1996). The structural repertoire of the human VKappa domainis also described by Tomlinson et al. EMBO J. 14:4628-4638 (1995), andthat of the VLambda domain by Williams et al. J. Mol. Biol., 264:220-232(1996). The contents of all these documents are to be incorporatedherein by reference.

L1 and L2 in the VL domain of an antibody with high human homology mayform a combination of predicted or actual canonical fold structures,which is identical to a combination of canonical fold structures knownto occur in a human germline VL domain. In non-limiting embodiments L1and L2 in the VLambda domain of an antibody with high human homology(e.g. an antibody containing a camelid-derived VL domain or a humanisedvariant thereof) may form one of the following canonical foldcombinations: 11-7, 13-7(A,B,C), 14-7(A,B), 12-11, 14-11 and 12-12 (asdefined in Williams et al. J. Mol. Biol. 264:220-32 (1996) and as shownon http://www.bioc.uzh.ch/antibody/Sequences/Germlines/VBase_hVL.html).In non-limiting embodiments L1 and L2 in the Vkappa domain may form oneof the following canonical fold combinations: 2-1, 3-1, 4-1 and 6-1 (asdefined in Tomlinson et al. EMBO J. 14:4628-38 (1995) and as shown onhttp://www.bioc.uzh.ch/antibody/Sequences/Germlines/VBase_hVK.html).

In a further embodiment, all three of L1, L2 and L3 in the VL domain ofan antibody with high human homology may exhibit a substantially humanstructure. It is preferred that the VL domain of the antibody with highhuman homology exhibits both high sequence identity/sequence homologywith human VL, and also that the hypervariable loops in the VL domainexhibit structural homology with human VL.

In one embodiment, the VL domain of a IL-22R antibody with high humanhomology may exhibit a sequence identity of 80% or greater, 85% orgreater, 90% or greater, 95% or greater, 97% or greater, or up to 99% oreven 100% with a human VL domain across the framework regions FR1, FR2,FR3 and FR4, and in addition hypervariable loop L1 and hypervariableloop L2 may form a combination of predicted or actual canonical foldstructures which is the same as a canonical fold combination known tooccur naturally in the same human VL domain.

It is, of course, envisaged that VH domains exhibiting high sequenceidentity/sequence homology with human VH, and also structural homologywith hypervariable loops of human VH will be combined with VL domainsexhibiting high sequence identity/sequence homology with human VL, andalso structural homology with hypervariable loops of human VL to provideantibodies with high human homology containing VH/VL pairings (e.gcamelid-derived VH/VL pairings) with maximal sequence and structuralhomology to human-encoded VH/VL pairings.

“scFv” or “scFv fragment”—An scFv or scFv fragment means a single chainvariable fragment. An scFv is a fusion protein of a VH domain and a VLdomain of an antibody connected via a linker.

B. IL-22R Antibodies

The present invention relates to antibodies and antigen bindingfragments thereof that specifically bind to human IL-22R. The IL-22Rantibodies and antibody fragments described herein possess properties,particularly combinations of properties, which are distinct and incertain cases superior, to the IL-22R antibodies of the prior art. Theseproperties and characteristics will now be described in further detail.

IL-22R Epitopes

The IL-22R antibodies and antigen binding fragments of the presentinvention bind to an epitope within the human IL-22R protein that doesnot include amino acid residue tyrosine 60 (Tyr60 or Y60), wherein thisamino acid position is defined with reference to the sequence of IL-22Rshown in FIG. 2 (SEQ ID NO: 71). This surface-exposed residue of IL-22Rhas previously been identified as critical to ligand binding, and asreported herein (see in particular Example 8) prior art IL-22R antibody280.346.TSY (as described in WO2011/061119) binds to an epitopeincluding this residue. The IL-22R antibodies and antigen bindingfragments of the present invention surprisingly bind to an epitope thatdoes not include this critical residue, and yet in certain embodiments,are capable of blocking IL-22 binding and/or inhibiting IL-22 and/orIL-20 mediated signaling via IL-22R.

The binding interface between the extracellular domain of IL-22R and itsligand IL-22 is characterised in Jones et al. (Structure 16(9):1333-1344 (2008)), the contents of which are incorporated herein intheir entirety. The extracellular domain of IL-22R consists of fiveloops (L2-L6), certain residues of which are critical for mediatinginteractions with surface-exposed residues on the IL-22 ligand. Tyrosine60 is located within the L2 loop formed by residues in the D1 domain ofthe IL-22R protein. In the IL-22/soluble IL-22R complex, residue Tyr60inserts into a small cavity on IL-22 created within site 1a of theligand. More specifically, this cavity is created at the intersection ofhelix F and the AB loop, two distinct secondary structures found in theIL-22 ligand. Helix F residues Lys162 and Glu166 on the ligand formhydrogen bonds to the OH of Tyr60 of the IL-22R and thus Tyr60 is a keyresidue mediating the interaction between site 1a on the ligand anddomain D1 of the receptor.

The antibodies and antigen binding fragments described herein bind to anepitope that does not include Tyr60. As noted above, Tyr60 is locatedwithin the L2 loop in the D1 domain of the receptor. In certainembodiments, the epitope to which the IL-22R antibodies or antigenbinding fragments bind is located at least in part in the D2 domain ofthe IL-22R i.e. between residues 125 and 228 of SEQ ID NO: 71. Incertain embodiments, the epitope to which the IL-22R antibodies orantigen binding fragments bind comprises at least one, at least two, atleast three amino acid residues located within the D2 domain of theIL-22R protein.

The inventors believe that the fact that the antibodies and antigenbinding fragments of the present invention bind to an epitope which doesnot include Tyr60 is relevant to their mode of action. The antibodiesand antigen binding fragments thereof are believed to interact with theD2 domain of IL22R, in lieu of or in addition to interacting with the D1domain. The D2 domain of IL-22R is known to be involved in theinteraction with the co-receptor. Without wishing to be bound by theory,the inventors believe that the antibody molecules of the presentinvention may have a dual mode of action, preventing binding to theligand and preventing interaction with the co-receptor.

The epitope to which the antibodies or antigen binding fragments of theinvention bind may be a linear epitope (i.e. an epitope consisting of alinear sequence of amino acids within the target antigen) or aconformational epitope (i.e. an epitope consisting of amino acids thatare not necessarily contiguous in the target antigen).

Binding Affinity

In certain embodiments, antibodies and antigen binding fragments of theinvention bind to human IL-22R with high affinity.

As used herein, the term “affinity” or “binding affinity” should beunderstood based on the usual meaning in the art in the context ofantibody binding, and reflects the strength and/or stability of bindingbetween an antigen and a binding site on an antibody or antigen bindingfragment thereof.

The binding affinity of an antibody or antigen binding fragment thereoffor its respective antigen can be determined experimentally usingtechniques known in the art. For example, BIACORE instruments measureaffinity based on the immobilization of a target protein or antigen on abiosensor chip while the antibody or antibody fragment is passed overthe immobilized target under specific flow conditions. These experimentsyield k_(on) and k_(off) measurements, which can be translated intoK_(D) values, wherein K_(D) is the equilibrium constant for thedissociation of an antigen with an antibody or fragment thereof. Thesmaller the K_(D) value, the stronger the binding interaction between anantibody and its target antigen. As noted above, the affinity of anantibody may be determined by Biacore, for example using the protocoldescribed elsewhere herein (see Example 6). The affinity of the antibodyor antigen binding fragment for human IL-22R, as measured by Biacore,may be determined using a recombinant full-length IL-22R construct, asdescribed for example, in Examples 3 and 6.

The IL-22R antibodies or antigen binding fragments thereof of theinvention may exhibit an off-rate (k_(off)) for IL-22R of less than2×10⁻³ s⁻¹, less than 1.5×10⁻³ s⁻¹, less than 1.2×10⁻³ s⁻¹ when testedas a mAb, for example when the affinity of a heavy chain variable domainpaired with a light chain variable domain is tested in the context of anIgG1 molecule. In preferred embodiments, the IL-22R antibodies orantigen binding fragments thereof of the invention exhibit an off-rate(k_(off)) for IL-22R of less than 2.5×10⁻⁴ s⁻¹, preferably less than1.5×10⁻⁴ s⁻¹, more preferably less than 1×10⁻⁴ s⁻¹ when tested as a mAb,for example when the affinity of a heavy chain variable domain pairedwith a light chain variable domain is tested in the context of an IgG1molecule.

The IL-22R antibodies or antigen binding fragments thereof of theinvention may exhibit an off-rate (k_(off)) for IL-22R in the range from1×10⁻⁶ s⁻¹ to 2×10⁻³ s⁻¹, preferably in the range from 1×10⁻⁶ s⁻¹ to2.5×10⁻⁴ s⁻¹, more preferably in the range from 1×10⁻⁵ s⁻¹ to 1×10⁻⁴s⁻¹.

The IL-22R antibodies or antigen binding fragments thereof of theinvention may exhibit a K_(D) value less than 3×10⁻⁹ M, less than2.5×10⁻⁹ M, less than 1×10⁻⁹ M. In preferred embodiments, the IL-22Rantibodies or antigen binding fragments thereof of the invention exhibita K_(D) value less than 5×10⁻¹⁰ M, preferably less than 2×10⁻¹⁰ M,preferably less than 1.5×10⁻¹⁰ M.

Inhibition of IL-22 and IL-20 Binding and Downstream Signalling

In certain embodiments, antibodies and antigen binding fragments of theinvention bind to human IL-22R and inhibit ligand binding to thisreceptor. This is particularly surprising given that the antibodiesdescribed herein bind to an epitope that does not include Tyr60. Asdescribed above, Tyr60 has been identified as a residue critical forligand binding and therefore the IL-22R antibodies and antigen bindingfragments of the present invention are surprising in their ability toinhibit ligand binding and in certain embodiments, inhibit signallingdownstream of IL-22R.

The antibodies and antigen binding fragments may bind human IL-22R andinhibit IL-22 binding or inhibit IL-20 binding. Preferably, theantibodies and antigen binding fragments described herein inhibit IL-22and IL-20 binding to the IL-22R.

Ligand binding to IL-22R on cells which express either the co-receptorIL-10R2 or IL-20R2 induces a conformational change in the receptorcomplex such that signalling pathways downstream of the receptor areactivated. The antibodies or antigen binding fragments thereof asdescribed herein may inhibit IL-22-dependent activation of IL-22R or mayinhibit IL-20-dependent activation of IL-22R. As used herein, the term“IL-22-dependent activation of IL-22R” should be taken to mean the chainof signalling events that occur downstream of the IL-22R receptorcomplex upon binding of the ligand IL-22 to the IL-22R/IL-10R2 complex.As used herein the term “IL-20-dependent activation of IL-22R” should betaken to mean the chain of signalling events that occur downstream ofthe IL-22R upon binding of the ligand IL-20 to the IL-22R/IL-20R2complex.

IL-22-dependent activation of IL-22R can be measured in cells or celllines expressing both the IL-22R and the co-receptor IL-10R2, forexample the BW-hIL-22R cell line described in Dumoutier et al. J BiolChem. 2009 Sep. 25; 284(39): 26377-84, the contents of which areincorporated herein in their entirety. IL-22 induces growth arrest inthe BW-hIL-22R cell line and therefore IL-22 dependent activation ofIL-22R can be determined in this cell line by measuring theproliferation of BW-hIL-22R cells, for example as described in Example6. In certain embodiments, the antibodies or antigen binding fragmentsdescribed herein exhibit an IC₅₀ (concentration at which 50% inhibitionof activation is achieved) in an IL-22 dependent cell-basedproliferation assay of less than 650 pM, preferably less than 600 pM.The cell-based proliferation assay is preferably an assay involving theBW-hIL-22R cell line as described herein.

IL-20-dependent activation of IL-22R can be measured in cells or celllines expressing both the IL-22R and the co-receptor IL-20R2, forexample the Baf3-hIL-22R/IL20Rb cell line described herein and also inDumoutier et al. J Biol Chem. 2009 Sep. 25; 284(39): 26377-84, thecontents of which are incorporated herein in their entirety. IL-20induces proliferation in the Baf3-hIL-22R/IL20Rb cell line and thereforeIL-20 dependent activation of IL-22R can be determined in this cell lineby measuring the proliferation of Baf3-hIL-22R/IL20Rb cells, for exampleas described in Example 6. In certain embodiments, the antibodies orantigen binding fragments described herein exhibit an IC₅₀(concentration at which 50% inhibition of activation is achieved) in anIL-20 dependent cell-based proliferation assay of less than 1 nM,preferably less than 800 pM. The cell-based proliferation assay ispreferably an assay involving the Baf3-hIL-22R/IL20Rb cell line asdescribed herein.

In addition to the proliferation assays described above, IL-22 and/orIL-20-dependent activation of IL-22R may be measured using alternativecellular assays, for example a STAT3 phosphorylation assay as describedin WO2011/061119.

In preferred embodiments, the antibodies or antigen binding fragments ofthe present invention bind to human IL-22R and inhibit both IL-22dependent activation of IL-22R and IL-20-dependent activation of IL-22R.In such embodiments, the inhibitory or “neutralising” activity of theantibodies or antigen binding fragments may be equipotent for both IL-22and IL-20-dependent activation. Alternatively, the inhibitory orneutralising activity of the antibodies or antigen binding fragments maybe greater for activation of IL-22R mediated by one of the ligands ascompared with activation of IL-22R mediated by the other ligand. Incertain embodiments, the antibodies or antigen binding fragments of theinvention will exhibit more potent inhibitory activity for IL-22dependent activation of IL-22R as compared with IL-20 dependentactivation of IL-22R. However, in such embodiments, the inhibitoryactivity of the antibody or antigen binding fragment for IL-22-dependentactivation of IL-22R will typically be less than 5 fold, preferably lessthan 4 fold greater, more preferably less than 2 fold greater than theinhibitory activity of the antibody or antigen binding fragment forIL-20-dependent activation of IL-22R.

Cross-Reactivity

In certain embodiments, the antibodies or antigen binding fragmentsdescribed herein that bind human IL-22R may cross-react with one or morespecies homologs of IL-22R, for example IL-22R homologs of primateorigin.

In certain embodiments, the antibodies or antigen binding fragments ofthe present invention do not cross-react with murine IL-22R.Alternatively or in addition, the antibodies or antigen bindingfragments may bind to one or more IL-22R homologs of primate origin, forexample IL-22R proteins from Rhesus and Cynomologus monkeys. Thecross-reactivity with other species homologs can be particularlyadvantageous in the development and testing of therapeutic antibodies.For example, pre-clinical toxicology testing of therapeutic antibodiesis frequently carried out in primate species including but not limitedto Rhesus and Cynomologus monkeys. Cross-reactivity with these specieshomologs can therefore be particularly advantageous for the developmentof antibodies as clinical candidates.

Camelid-Derived Antibodies

In certain embodiments of the invention, the antibodies or antigenbinding fragments thereof described herein may comprise at least onehypervariable loop or complementarity determining region obtained from aVH domain or a VL domain of a species in the family Camelidae. Inparticular, the antibody or antigen binding fragment may comprise VHand/or VL domains, or CDRs thereof, obtained by active immunisation ofoutbred camelids, e.g. llamas, with an IL-22R antigen or fragmentthereof.

By “hypervariable loop or complementarity determining region obtainedfrom a VH domain or a VL domain of a species in the family Camelidae” ismeant that that hypervariable loop (HV) or CDR has an amino acidsequence which is identical, or substantially identical, to the aminoacid sequence of a hypervariable loop or CDR which is encoded by aCamelidae immunoglobulin gene. In this context “immunoglobulin gene”includes germline genes, immunoglobulin genes which have undergonerearrangement, and also somatically mutated genes. Thus, the amino acidsequence of the HV or CDR obtained from a VH or VL domain of a Camelidaespecies may be identical to the amino acid sequence of a HV or CDRpresent in a mature Camelidae conventional antibody. The term “obtainedfrom” in this context implies a structural relationship, in the sensethat the HVs or CDRs of the IL-22R antibody embody an amino acidsequence (or minor variants thereof) which was originally encoded by aCamelidae immunoglobulin gene. However, this does not necessarily implya particular relationship in terms of the production process used toprepare the IL-22R antibody.

Camelid-derived IL-22R antibodies may be derived from any camelidspecies, including inter alia, llama, dromedary, alpaca, vicuna, guanacoor camel.

IL-22R antibodies comprising camelid-derived VH and VL domains, or CDRsthereof, are typically recombinantly expressed polypeptides, and may bechimeric polypeptides. The term “chimeric polypeptide” refers to anartificial (non-naturally occurring) polypeptide which is created byjuxtaposition of two or more peptide fragments which do not otherwiseoccur contiguously. Included within this definition are “species”chimeric polypeptides created by juxtaposition of peptide fragmentsencoded by two or more species, e.g. camelid and human.

Camelid-derived CDRs may comprise one of the CDR sequences shown as SEQID NOs: 6 and 13 (heavy chain CDR3), or SEQ ID NOs: 4 and 11 (heavychain CDR2) or SEQ ID NOs: 2 and 9 (heavy chain CDR1) or one of the CDRsequences shown as SEQ ID NOs: 20 and 27 (light chain CDR3), or SEQ IDNOs: 18, 25 and 47 (light chain CDR2) or SEQ ID NOs: 16 and 23 (lightchain CDR1).

In one embodiment the entire VH domain and/or the entire VL domain maybe obtained from a species in the family Camelidae. In specificembodiments, the camelid-derived VH domain may comprise an amino acidsequence selected from SEQ ID NOs: 29 and 31, whereas thecamelid-derived VL domain may comprise an amino acid sequence selectedfrom SEQ ID NOs: 30, 32 and 62. The camelid-derived VH domain and/or thecamelid-derived VL domain may then be subject to protein engineering, inwhich one or more amino acid substitutions, insertions or deletions areintroduced into the camelid amino acid sequence. These engineeredchanges preferably include amino acid substitutions relative to thecamelid sequence. Such changes include “humanisation” or “germlining”wherein one or more amino acid residues in a camelid-encoded VH or VLdomain are replaced with equivalent residues from a homologoushuman-encoded VH or VL domain. In certain embodiments, thecamelid-derived VH domain may exhibit at least 90%, 95%, 97%, 98% or 99%identity with the amino acid sequence shown as SEQ ID NOs: 29 or 31.Alternatively, or in addition, the camelid-derived VL domain may exhibitat least 90%, 95%, 97%, 98% or 99% identity with the amino acid sequenceshown as SEQ ID NOs: 30, 32 or 62.

Isolated camelid VH and VL domains obtained by active immunisation of acamelid (e.g. llama) with a human IL-22R antigen can be used as a basisfor engineering antigen binding polypeptides according to the invention.Starting from intact camelid VH and VL domains, it is possible toengineer one or more amino acid substitutions, insertions or deletionswhich depart from the starting camelid sequence. In certain embodiments,such substitutions, insertions or deletions may be present in theframework regions of the VH domain and/or the VL domain. The purpose ofsuch changes in primary amino acid sequence may be to reduce presumablyunfavourable properties (e.g. immunogenicity in a human host (so-calledhumanization), sites of potential product heterogeneity and orinstability (glycosylation, deamidation, isomerisation, etc.) or toenhance some other favourable property of the molecule (e.g. solubility,stability, bioavailability etc.). In other embodiments, changes inprimary amino acid sequence can be engineered in one or more of thehypervariable loops (or CDRs) of a Camelidae VH and/or VL domainobtained by active immunisation. Such changes may be introduced in orderto enhance antigen binding affinity and/or specificity, or to reducepresumably unfavourable properties, e.g. immunogenicity in a human host(so-called humanization), sites of potential product heterogeneity andor instability, glycosylation, deamidation, isomerisation, etc., or toenhance some other favourable property of the molecule, e.g. solubility,stability, bioavailability, etc.

Thus, in one embodiment, the invention provides a variant IL-22Rantibody which contains at least one amino acid substitution in at leastone framework or CDR region of either the VH domain or the VL domain incomparison to a camelid-derived VH or VL domain, examples of whichinclude but are not limited to the camelid VH domains comprising theamino acid sequences shown as SEQ ID NOs: 29 and 31, and the camelid VLdomains comprising the amino acid sequences shown as SEQ ID NOs: 30, 32or 62.

In other embodiments, there are provided “chimeric” antibody moleculescomprising camelid-derived VH and VL domains (or engineered variantsthereof) and one or more constant domains from a non-camelid antibody,for example human-encoded constant domains (or engineered variantsthereof). In such embodiments it is preferred that both the VH domainand the VL domain are obtained from the same species of camelid, forexample both VH and VL may be from Lama glama or both VH and VL may befrom Lama pacos (prior to introduction of engineered amino acid sequencevariation). In such embodiments both the VH and the VL domain may bederived from a single animal, particularly a single animal which hasbeen actively immunised with an IL-22R antigen.

As an alternative to engineering changes in the primary amino acidsequence of Camelidae VH and/or VL domains, individual camelid-derivedhypervariable loops or CDRs, or combinations thereof, can be isolatedfrom camelid VH/VL domains and transferred to an alternative (i.e.non-Camelidae) framework, e.g. a human VH/VL framework, by CDR grafting.In particular, non-limiting, embodiments the camelid-derived CDRs may beselected from CDRs having the amino acid sequences shown as SEQ ID NOs:6 and 13 (heavy chain CDR3), or SEQ ID NOs: 4 and 11 (heavy chain CDR2)or SEQ ID NOs: 2 and 9 (heavy chain CDR1) or one of the CDR sequencesshown as SEQ ID NOs: 20 and 27 (light chain CDR3), or SEQ ID NOs: 18, 25and 47 (light chain CDR2) or SEQ ID NOs:16 and 23 (light chain CDR1).

IL-22R antibodies comprising camelid-derived VH and VL domains, or CDRsthereof, can take various different embodiments in which both a VHdomain and a VL domain are present. The term “antibody” herein is usedin the broadest sense and encompasses, but is not limited to, monoclonalantibodies (including full length monoclonal antibodies), polyclonalantibodies, multispecific antibodies (e.g., bispecific antibodies), solong as they exhibit the appropriate immunological specificity for anIL-22R protein. The term “monoclonal antibody” as used herein refers toan antibody obtained from a population of substantially homogeneousantibodies, i.e., the individual antibodies comprising the populationare identical except for possible naturally occurring mutations that maybe present in minor amounts. Monoclonal antibodies are highly specific,being directed against a single antigenic site. Furthermore, in contrastto conventional (polyclonal) antibody preparations which typicallyinclude different antibodies directed against different determinants(epitopes) on the antigen, each monoclonal antibody is directed againsta single determinant or epitope on the antigen.

“Antibody fragments” comprise a portion of a full length antibody,generally the antigen binding or variable domain thereof. Examples ofantibody fragments include Fab, Fab′, F(ab′)2, bi-specific Fab's, and Fvfragments, diabodies, linear antibodies, single-chain antibodymolecules, a single chain variable fragment (scFv) and multispecificantibodies formed from antibody fragments (see Holliger and Hudson,Nature Biotechnol. 23:1126-36 (2005), the contents of which areincorporated herein by reference).

In non-limiting embodiments, IL-22R antibodies comprisingcamelid-derived VH and VL domains, or CDRs thereof, may comprise CH1domains and/or CL domains, the amino acid sequence of which is fully orsubstantially human. Where the antigen binding polypeptide of theinvention is an antibody intended for human therapeutic use, it istypical for the entire constant region of the antibody, or at least apart thereof, to have fully or substantially human amino acid sequence.Therefore, one or more or any combination of the CH1 domain, hingeregion, CH2 domain, CH3 domain and CL domain (and CH4 domain if present)may be fully or substantially human with respect to its amino acidsequence.

Advantageously, the CH1 domain, hinge region, CH2 domain, CH3 domain andCL domain (and CH4 domain if present) may all have fully orsubstantially human amino acid sequence. In the context of the constantregion of a humanised or chimeric antibody, or an antibody fragment, theterm “substantially human” refers to an amino acid sequence identity ofat least 90%, or at least 92%, or at least 95%, or at least 97%, or atleast 99% with a human constant region. The term “human amino acidsequence” in this context refers to an amino acid sequence which isencoded by a human immunoglobulin gene, which includes germline,rearranged and somatically mutated genes. The invention alsocontemplates polypeptides comprising constant domains of “human”sequence which have been altered, by one or more amino acid additions,deletions or substitutions with respect to the human sequence, exceptingthose embodiments where the presence of a “fully human” hinge region isexpressly required.

The presence of a “fully human” hinge region in the IL-22R antibodies ofthe invention may be beneficial both to minimise immunogenicity and tooptimise stability of the antibody. As discussed elsewhere herein, it iscontemplated that one or more amino acid substitutions, insertions ordeletions may be made within the constant region of the heavy and/or thelight chain, particularly within the Fc region. Amino acid substitutionsmay result in replacement of the substituted amino acid with a differentnaturally occurring amino acid, or with a non-natural or modified aminoacid. Other structural modifications are also permitted, such as forexample changes in glycosylation pattern (e.g. by addition or deletionof N- or O-linked glycosylation sites). Depending on the intended use ofthe antibody, it may be desirable to modify the antibody of theinvention with respect to its binding properties to Fc receptors, forexample to modulate effector function. For example cysteine residue(s)may be introduced in the Fc region, thereby allowing interchaindisulfide bond formation in this region. The homodimeric antibody thusgenerated may have improved internalization capability and/or increasedcomplement-mediated cell killing and antibody-dependent cellularcytotoxicity (ADCC). See Caron et al., J. Exp. Med. 176:1191-1195 (1992)and Shopes, B. J. Immunol. 148:2918-2922 (1992). The invention alsocontemplates immunoconjugates comprising an antibody as described hereinconjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin(e.g., an enzymatically active toxin of bacterial, fungal, plant oranimal origin, or fragments thereof), or a radioactive isotope (i.e., aradioconjugate). Fc regions may also be engineered for half-lifeextension, as described by Chan and Carter, Nature Reviews: Immunology,Vol. 10, pp 301-316, 2010, incorporated herein by reference.

In yet another embodiment, the Fc region is modified to increase theability of the antibody to mediate antibody dependent cellularcytotoxicity (ADCC) and/or to increase the affinity of the antibody foran Fcγ receptor by modifying one or more amino acids. In alternativeembodiments, the Fc region may be engineered such that there is noeffector function. An IL-22R antibody having no Fc effector function maybe particularly useful as a receptor blocking agent. In certainembodiments, the antibodies of the invention may have an Fc regionderived from naturally-occurring IgG isotypes having reduced effectorfunction, for example IgG4. Fc regions derived from IgG4 may be furthermodified to increase therapeutic utility, for example by theintroduction of modifications that minimise the exchange of arms betweenIgG4 molecules in vivo.

In still another embodiment, the glycosylation of an antibody ismodified. For example, an aglycosylated antibody can be made (i.e., theantibody lacks glycosylation). Glycosylation can be altered to, forexample, increase the affinity of the antibody for the target antigen.Such carbohydrate modifications can be accomplished by; for example,altering one or more sites of glycosylation within the antibodysequence. For example, one or more amino acid substitutions can be madethat result in elimination of one or more variable region frameworkglycosylation sites to thereby eliminate glycosylation at that site.Such aglycosylation may increase the affinity of the antibody forantigen.

Also envisaged are variant IL-22R antibodies having an altered type ofglycosylation, such as a hypofucosylated antibody having reduced amountsof fucosyl residues or a fully or partially de-fucosylated antibody (asdescribed by Natsume et al., Drug Design Development and Therapy, Vol.3, pp 7-16, 2009) or an antibody having increased bisecting GlcNacstructures. Such altered glycosylation patterns have been demonstratedto increase the ADCC activity of antibodies, producing typically 10-foldenhancement of ADCC relative to an equivalent antibody comprising a“native” human Fc domain. Such carbohydrate modifications can beaccomplished by, for example, expressing the antibody in a host cellwith altered glycosylation enzymatic machinery (as described byYamane-Ohnuki and Satoh, mAbs 1:3, 230-236, 2009). Examples ofnon-fucosylated antibodies with enhanced ADCC function are thoseproduced using the Potelligent™ technology of BioWa Inc.

The invention can, in certain embodiments, encompass chimericCamelidae/human antibodies, and in particular chimeric antibodies inwhich the VH and VL domains are of fully camelid sequence (e.g. Llama oralpaca) and the remainder of the antibody is of fully human sequence.IL-22R antibodies can include antibodies comprising “humanised” or“germlined” variants of camelid-derived VH and VL domains, or CDRsthereof, and camelid/human chimeric antibodies, in which the VH and VLdomains contain one or more amino acid substitutions in the frameworkregions in comparison to camelid VH and VL domains obtained by activeimmunisation of a camelid with an IL-22R antigen or a fragment thereof.Such “humanisation” increases the % sequence identity with humangermline VH or VL domains by replacing mis-matched amino acid residuesin a starting Camelidae VH or VL domain with the equivalent residuefound in a human germline-encoded VH or VL domain. IL-22R antibodies mayalso be CDR-grafted antibodies in which CDRs (or hypervariable loops)derived from a camelid antibody, or otherwise encoded by a camelid gene,are grafted onto a human VH and VL framework, with the remainder of theantibody also being of fully human origin. Such CDR-grafted IL-22Rantibodies may contain CDRs having the amino acid sequences shown as SEQID NOs: 6 and 13 (heavy chain CDR3), or SEQ ID NOs: 4 and 11 (heavychain CDR2) or SEQ ID NOs: 2 and 9 (heavy chain CDR1) or one of the CDRsequences shown as SEQ ID NOs: 20 and 27 (light chain CDR3), or SEQ IDNOs: 18, 25 and 47 (light chain CDR2) or SEQ ID NOs:16 and 23 (lightchain CDR1).

Humanised, chimeric and CDR-grafted IL-22R antibodies as describedabove, particularly antibodies comprising hypervariable loops or CDRsderived from active immunisation of camelids, can be readily producedusing conventional recombinant DNA manipulation and expressiontechniques, making use of prokaryotic and eukaryotic host cellsengineered to produce the polypeptide of interest and including but notlimited to bacterial cells, yeast cells, mammalian cells, insect cells,plant cells, some of them as described herein and illustrated in theaccompanying examples.

Camelid-derived IL-22R antibodies include variants wherein thehypervariable loop(s) or CDR(s) of the VH domain and/or the VL domainare obtained from a conventional camelid antibody raised against humanIL-22R, but wherein at least one of said (camelid-derived) hypervariableloops or CDRs has been engineered to include one or more amino acidsubstitutions, additions or deletions relative to the camelid-encodedsequence. Such changes include “humanisation” of the hypervariableloops/CDRs. Camelid-derived HVs/CDRs which have been engineered in thismanner may still exhibit an amino acid sequence which is “substantiallyidentical” to the amino acid sequence of a camelid-encoded HV/CDR. Inthis context, “substantial identity” may permit no more than one, or nomore than two amino acid sequence mis-matches with the camelid-encodedHV/CDR. Particular embodiments of the IL-22R antibody may containhumanised variants of the CDR sequences shown as SEQ ID NOs: 6 and 13(heavy chain CDR3), or SEQ ID NOs: 4 and 11 (heavy chain CDR2) or SEQ IDNOs: 2 and 9 (heavy chain CDR1) or one of the CDR sequences shown as SEQID NOs: 20 and 27 (light chain CDR3), or SEQ ID NOs: 18, 25 and 47(light chain CDR2) or SEQ ID NOs:16 and 23 (light chain CDR1).

The camelid-derived IL-22R antibodies provided herein may be of anyisotype. Antibodies intended for human therapeutic use will typically beof the IgA, IgD, IgE, IgG, IgM type, often of the IgG type, in whichcase they can belong to any of the four sub-classes IgG1, IgG2a and b,IgG3 or IgG4. Within each of these sub-classes it is permitted to makeone or more amino acid substitutions, insertions or deletions within theFc portion, or to make other structural modifications, for example toenhance or reduce Fc-dependent functionalities.

Preferred IL-22R Antibodies—230C9 and 223G5 and Antibodies RelatedThereto

Preferred IL-22R antibodies and antigen binding fragments thereofaccording to the present invention are humanised variants and germlinedvariants of the camelid-derived antibodies described herein. Humanisedand germlined variants exhibit high human homology, and preferablyexhibit high homology to human IgG molecules, more preferably IgG1. Incertain embodiments, preferred IL-22R antibodies and antigen bindingfragments according to the present invention comprise hypervariableloops or CDRs having human or human-like canonical folds, as describedelsewhere herein.

The preferred IL-22R antibodies according to the present invention mayexhibit an amino acid sequence identity of 93% or greater with one ormore human VH domains, particularly across the framework regions FR1,FR2, FR3 and FR4. Alternatively or in addition, the preferred IL-22Rantibodies may exhibit an amino acid sequence identity of 96% or greaterwith one or more human VL domains, particularly across the frameworkregions FR1, FR2, FR3 and FR4. The preferred IL-22R antibodies accordingto the present invention may exhibit an amino acid sequence identity of93% or greater with one or more human VH domains, and a combined aminoacid sequence identity of 93% or greater with one or more human VHdomains and one or more human VL domains, particularly where sequenceidentity is determined across the framework regions.

Due to the high human homology, the preferred IL-22R antibodies providedherein exhibit low immunogenicity, as assessed by the Ionza's Epibase™platform (DRB-1 score) using the “HLA class II-Caucasian v3.0” settings.In certain embodiments, the IL-22R antibodies of the invention exhibit aDRB-1 score of less than 950, preferably less than 850, more preferablyless than 750, most preferably less than 650.

Preferred IL-22R antibodies of the invention are monoclonal antibodiescontaining a hinge region, a CH2 domain and a CH3 domain from a humanIgG, preferably an IgG1. In certain embodiments, the Fc region of themonoclonal antibody has no effector function i.e. is a null Fc. This isparticularly useful for therapeutic blocking antibodies.

The preferred IL-22R antibodies and antigen binding fragments of theinvention exhibit a combination of properties that render them superiorto IL-22R antibodies described in the prior art. Preferred IL-22Rantibodies and antigen binding fragments of the invention may exhibitthe following combination of properties:

-   -   (i) binding to an epitope within the IL-22R protein that does        not include Tyr60;    -   (ii) binding to an epitope that is located at least in part in        the D2 domain of the IL-22R protein;    -   (iii) high binding affinity to human IL-22R;    -   (iv) inhibition of IL-22-dependent activation of IL-22R and        IL-20-dependent activation of IL-22R;    -   (v) no cross-reactivity with murine IL-22R; and    -   (vi) cross-reactivity with Rhesus and/or Cynomologus IL-22R.

The preferred IL-22R antibodies of the invention may bind to an epitopethat does not include the critical residue Tyr60. The preferred IL-22Rantibodies of the invention may bind to an epitope that is located atleast in part in the D2 domain of IL-22R, wherein the D2 domain is fromamino acid 125 to amino acid 228 of SEQ ID NO: 71. The preferred IL-22Rantibodies of the invention may also bind to human IL-22R with highaffinity, typically exhibiting an off-rate (wherein k_(off) is measuredby Biacore) for human IL-22R of less than 2.5×10⁻⁴ s⁻¹ when measured asa mAb. In certain embodiments, the preferred IL-22R antibodies of theinvention bind to human IL-22R with high affinity exhibiting an off-rate(wherein k_(off) is measured by Biacore) for human IL-22R in the range1×10⁻⁶ s⁻¹ to 2.5×10⁻⁴ s⁻¹. In certain embodiments, the preferred IL-22Rantibodies of the invention bind to human IL-22R exhibiting a K_(D)value less than 5×10⁻¹⁰ M.

The preferred IL-22R antibodies may also inhibit both IL-22 and IL-20dependent activation of IL-22R, and typically exhibit an inhibitoryactivity for IL-20-dependent activation that is less than four timesgreater than the inhibitory activity displayed for IL-22-dependentactivation. The preferred IL-22R antibodies may bind to human IL-22Rwith high affinity and cross-react with IL-22R species homologues fromRhesus and Cynomologus monkeys, but do not cross-react with murineIL-22R.

In certain embodiments, the preferred antibodies and antigen bindingfragments according to the present invention comprise at least one heavychain variable domain (VH) and at least one light chain variable domain(VL) wherein the VH domain comprises:

-   -   a variable heavy chain CDR3 comprising or consisting of the        amino acid sequence [VGFSGTYYSES], SEQ ID NO: 6,    -   a variable heavy chain CDR2 comprising or consisting of the        amino acid sequence [SIYNDASNTAYSDSVKG], SEQ ID NO: 36,    -   a variable heavy chain CDR1 comprising or consisting of the        amino acid sequence [SYDMN], SEQ ID NO: 34,        and the VL domain comprises:    -   a variable light chain CDR3 comprising or consisting of the        amino acid sequence [QSGSSSSNAV], SEQ ID NO: 54,    -   a variable light chain CDR2 comprising or consisting of the        amino acid sequence [GQNNRPS], SEQ ID NO. 47,    -   a variable light chain CDR1 comprising or consisting of the        amino acid sequence [QGGYYAH], SEQ ID NO: 16.

The antibodies and antigen binding fragments having the VH and VL domainCDR sequences as defined above may comprise a VH domain comprising orconsisting of the sequence of SEQ ID NO:63 and/or a VL domain comprisingor consisting of the sequence of SEQ ID NO:64. In certain embodiments,provided herein are antibodies or antigen binding fragments thereof,comprising a heavy chain variable domain and a light chain variabledomain, the heavy chain variable domain comprising a VH sequence with atleast 85% sequence identity, or at least 90% sequence identity, or atleast 95% sequence identity, or at least 97%, 98% or 99% sequenceidentity, to the amino acid sequence shown as SEQ ID NO:63 and/or thelight chain variable domain comprising a VL with at least 85% sequenceidentity, or at least 90% sequence identity, or at least 95% sequenceidentity, or at least 97%, 98% or 99% sequence identity, to the aminoacid sequence shown as SEQ ID NO:64. For embodiments wherein the domainsof the antibodies or antigen binding fragments are defined by aparticular percentage sequence identity to a reference sequence, the VHand/or VL domains may retain identical CDR sequences to those present inthe reference sequence such that the variation is present only withinthe framework regions.

The antibodies and antigen binding fragments having the VH and VL domainCDR sequences as defined above may comprise a full-length immunoglobulinheavy chain comprising or consisting of the amino acid sequence of SEQID NO: 67 and/or a full length immunoglobulin light chain comprising orconsisting of the amino acid sequence of SEQ ID NO: 68. In certainembodiments, provided herein are antibodies comprising a heavy chainwith at least 85% sequence identity, or at least 90% sequence identity,or at least 95% sequence identity, or at least 97%, 98% or 99% sequenceidentity, to the amino acid sequence shown as SEQ ID NO:67 and/or alight chain with at least 85% sequence identity, or at least 90%sequence identity, or at least 95% sequence identity, or at least 97%,98% or 99% sequence identity, to the amino acid sequence shown as SEQ IDNO:68. For embodiments wherein the chains of the antibodies are definedby a particular percentage sequence identity to a reference sequence,the heavy chain and/or light chain may retain identical CDR sequences tothose present in the reference sequence such that the variation ispresent only outside the CDR regions.

In certain embodiments, the preferred antibodies and antigen bindingfragments according to the present invention comprise at least one heavychain variable domain (VH) and at least one light chain variable domain(VL) wherein the VH domain comprises:

-   -   a variable heavy chain CDR3 comprising or consisting of the        amino acid sequence [PPGPFKAHYNGAKY], SEQ ID NO: 43,    -   a variable heavy chain CDR2 comprising or consisting of the        amino acid sequence [GIHISGGITYYTDSVKG], SEQ ID NO: 41,    -   a variable heavy chain CDR1 comprising or consisting of the        amino acid sequence [SYFMS], SEQ ID NO: 9,        and the VL domain comprises:    -   a variable light chain CDR3 comprising or consisting of the        amino acid sequence [ASYRLYADYV], SEQ ID NO: 27,    -   a variable light chain CDR2 comprising or consisting of the        amino acid sequence [EVNKRSS], SEQ ID NO. 59,    -   a variable light chain CDR1 comprising or consisting of the        amino acid sequence [TGTSSDIGSYNYVS], SEQ ID NO: 57.

The antibodies and antigen binding fragments having the VH and VL domainCDR sequences as defined above may comprise a VH domain comprising orconsisting of the sequence of SEQ ID NO:65 and/or a VL domain comprisingor consisting of the sequence of SEQ ID NO:66. In certain embodiments,provided herein are antibodies or antigen binding fragments thereof,comprising a heavy chain variable domain and a light chain variabledomain, the heavy chain variable domain comprising a VH sequence with atleast 85% sequence identity, or at least 90% sequence identity, or atleast 95% sequence identity, or at least 97%, 98% or 99% sequenceidentity, to the amino acid sequence shown as SEQ ID NO:65 and/or thelight chain variable domain comprising a VL with at least 85% sequenceidentity, or at least 90% sequence identity, or at least 95% sequenceidentity, or at least 97%, 98% or 99% sequence identity, to the aminoacid sequence shown as SEQ ID NO:66. For embodiments wherein the domainsof the antibodies or antigen binding fragments are defined by aparticular percentage sequence identity to a reference sequence, the VHand/or VL domains may retain identical CDR sequences to those present inthe reference sequence such that the variation is present only withinthe framework regions.

The antibodies and antigen binding fragments having the VH and VL domainCDR sequences as defined above may comprise a full-length immunoglobulinheavy chain comprising or consisting of the amino acid sequence of SEQID NO: 69 and/or a full length immunoglobulin light chain comprising orconsisting of the amino acid sequence of SEQ ID NO: 70. In certainembodiments, provided herein are antibodies comprising a heavy chainwith at least 85% sequence identity, or at least 90% sequence identity,or at least 95% sequence identity, or at least 97%, 98% or 99% sequenceidentity, to the amino acid sequence shown as SEQ ID NO:69 and/or alight chain with at least 85% sequence identity, or at least 90%sequence identity, or at least 95% sequence identity, or at least 97%,98% or 99% sequence identity, to the amino acid sequence shown as SEQ IDNO:70. For embodiments wherein the chains of the antibodies are definedby a particular percentage sequence identity to a reference sequence,the heavy chain and/or light chain may retain identical CDR sequences tothose present in the reference sequence such that the variation ispresent only outside the CDR regions.

Cross-Competing Antibodies

The present invention also includes (monoclonal) antibodies orantigen-binding fragments thereof that “cross-compete” with theantibodies or antigen binding fragments disclosed herein.

In particular, provided herein are antibodies or antigen-bindingfragments thereof, which bind to the cytokine receptor IL-22R andcross-compete with antibodies or antigen binding fragments thereof,comprising a combination of variable heavy chain CDR3 (HCDR3), variableheavy chain CDR2 (HCDR2) and variable heavy chain CDR1 (HCDR1), variablelight chain CDR3 (LCDR3), variable light chain CDR2 (LCDR2) and variablelight chain CDR1 (LCDR1) wherein the combination is selected from thegroup consisting of:

(i) HCDR3 comprising SEQ ID NO: 43; HCDR2 comprising SEQ ID NO: 41;HCDR1 comprising SEQ ID NO: 9; LCDR3 comprising SEQ ID NO: 27; LCDR2comprising SEQ ID NO: 59; LCDR1 comprising SEQ ID NO: 57; and

(ii) HCDR3 comprising SEQ ID NO: 13; HCDR2 comprising SEQ ID NO: 11;HCDR1 comprising SEQ ID NO: 9; LCDR3 comprising SEQ ID NO: 27; LCDR2comprising SEQ ID NO: 25; LCDR1 comprising SEQ ID NO: 23.

In certain embodiments, provided herein are antibodies orantigen-binding fragments thereof, which bind to the cytokine receptorIL-22R and cross-compete with antibodies or antigen binding fragmentsthereof comprising at least one heavy chain variable domain (VH) and atleast one light chain variable domain (VL) wherein the VH domaincomprises:

-   -   a variable heavy chain CDR3 comprising or consisting of the        amino acid sequence [PPGPFKAHYNGAKY], SEQ ID NO: 43,    -   a variable heavy chain CDR2 comprising or consisting of the        amino acid sequence [GIHISGGITYYTDSVKG], SEQ ID NO: 41,    -   a variable heavy chain CDR1 comprising or consisting of the        amino acid sequence [SYFMS], SEQ ID NO: 9,        and the VL domain comprises:    -   a variable light chain CDR3 comprising or consisting of the        amino acid sequence [ASYRLYADYV], SEQ ID NO: 27,    -   a variable light chain CDR2 comprising or consisting of the        amino acid sequence [EVNKRSS], SEQ ID NO. 59,    -   a variable light chain CDR1 comprising or consisting of the        amino acid sequence [TGTSSDIGSYNYVS], SEQ ID NO: 57.

The cross-competing antibodies or antigen-binding fragments thereof maycompete with antibodies or antigen binding fragments having a VH domaincomprising or consisting of the sequence of SEQ ID NO:65 and/or a VLdomain comprising or consisting of the sequence of SEQ ID NO:66. Thecross-competing antibodies or antigen-binding fragments thereof maycompete with antibodies having a full-length immunoglobulin heavy chaincomprising or consisting of the amino acid sequence of SEQ ID NO: 69and/or a full length immunoglobulin light chain comprising or consistingof the amino acid sequence of SEQ ID NO: 70.

In the context of the present invention, cross-competing antibodies arethose that bind IL-22R at site(s) that overlap or are identical to thesite(s) at which the present IL-22R antibodies bind. Competing(monoclonal) antibodies or antigen-binding fragments thereof can beidentified, for example, via an antibody competition assay. For example,an IL-22R antigen or fragment thereof can be bound to a solid support.Then, an antibody or antigen binding fragment thereof of the presentinvention and a monoclonal antibody or antigen-binding fragment thereofsuspected of being able to compete with such invention antibody areadded. One of the two molecules is labelled. If the labelled compoundand the unlabeled compound bind to separate and discrete sites on theIL-22R antigen, the labelled compound will bind to the same levelwhether or not the suspected competing compound is present. However, ifthe sites of interaction are identical (or overlapping), the unlabeledcompound will compete, and the amount of labelled compound bound to theantigen will be lowered. If the unlabeled compound is present in excess,very little, if any, labelled compound will bind. For purposes of thepresent invention, competing monoclonal antibodies or antigen-bindingfragments thereof are those that decrease the binding of the presentantibodies to IL-22R by about 50%, about 60%, about 70%, about 80%,about 85%, about 90%, about 95%, or about 99%. Details of procedures forcarrying out such competition assays are well known in the art and canbe found, for example, in Harlow and Lane (1988) Antibodies, ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., pages 567-569, ISBN 0-87969-314-2. Such assays can be madequantitative by using purified antibodies. A standard curve isestablished by titrating one antibody against itself, i.e., the sameantibody is used for both the label and the competitor. The capacity ofan unlabeled competing monoclonal antibody or antigen-binding fragmentthereof to inhibit the binding of the labelled molecule to the plate istitrated. The results are plotted, and the concentrations necessary toachieve the desired degree of binding inhibition are compared.

Polynucleotides Encoding IL-22R Antibodies

The invention also provides polynucleotide molecules encoding the IL-22Rantibodies of the invention or fragments thereof, also expressionvectors containing said nucleotide sequences of the invention operablylinked to regulatory sequences which permit expression of the antibodiesor fragments thereof in a host cell or cell-free expression system, anda host cell or cell-free expression system containing this expressionvector.

In particular embodiments, the polynucleotide encoding the IL-22Rantibody of the invention may comprise one or more of the polynucleotidesequences shown as SEQ ID NOs: 52, 73, 74, 75, 76, 77, 78, 79, 80 or 81,which sequences encode VH or VL domains of IL-22R antibodies.

In certain embodiments, the polynucleotide encoding the IL-22R antibodyof the invention may comprise a variant sequence which encodes afunctional VH or VL domain of an IL-22R antibody, wherein said variantsequence exhibits at least 80%, 85%, 90%, 95%, 97% or 99% sequenceidentity when optimally aligned to any one of SEQ ID NOs: 52, 73, 74,75, 76, 77, 78, 79, 80 or 81.

In this context, % sequence identity between two polynucleotidesequences may be determined by comparing these two sequences aligned inan optimum manner and in which the polynucleotide sequence to becompared can comprise additions or deletions with respect to thereference sequence for an optimum alignment between these two sequences.The percentage of identity is calculated by determining the number ofidentical positions for which the nucleotide residue is identicalbetween the two sequences, by dividing this number of identicalpositions by the total number of positions in the comparison window andby multiplying the result obtained by 100 in order to obtain thepercentage of identity between these two sequences. For example, it ispossible to use the BLAST program, “BLAST 2 sequences” (Tatusova et al,“Blast 2 sequences—a new tool for comparing protein and nucleotidesequences”, FEMS Microbiol Lett. 174:247-250) available on the sitehttp://www.ncbi.nlm.nih.gov/gorf/bl2.html, the parameters used beingthose given by default (in particular for the parameters “open gappenalty”: 5, and “extension gap penalty”: 2; the matrix chosen being,for example, the matrix “BLOSUM 62” proposed by the program), thepercentage of identity between the two sequences to be compared beingcalculated directly by the program.

In certain embodiments, the heavy chain variable domain and the lightchain variable domain of the IL-22R antibodies or antigen-bindingfragments according to the present invention are encoded by acombination of first and second polynucleotide sequences, wherein thefirst and second polynucleotide sequences are selected from thefollowing pairs:

(i) a first polynucleotide encoding a variable heavy chain domaincomprising SEQ ID NO: 52 and a second polynucleotide encoding a variablelight chain domain comprising SEQ ID NO: 73;

(ii) a first polynucleotide encoding a variable heavy chain domaincomprising SEQ ID NO: 74 and a second polynucleotide encoding a variablelight chain domain comprising SEQ ID NO: 75;

(iii) a first polynucleotide encoding a variable heavy chain domaincomprising SEQ ID NO: 76 and a second polynucleotide encoding a variablelight chain domain comprising SEQ ID NO: 77;

(iv) a first polynucleotide encoding a variable heavy chain domaincomprising SEQ ID NO: 78 and a second polynucleotide encoding a variablelight chain domain comprising SEQ ID NO: 79; or

(v) a first polynucleotide encoding a variable heavy chain domaincomprising SEQ ID NO: 80 and a second polynucleotide encoding a variablelight chain domain comprising SEQ ID NO: 81.

Polynucleotide molecules encoding the antibodies of the inventioninclude, for example, recombinant DNA molecules. The terms “nucleicacid”, “polynucleotide” or a “polynucleotide molecule” as used hereininterchangeably and refer to any DNA or RNA molecule, either single- ordouble-stranded and, if single-stranded, the molecule of itscomplementary sequence. In discussing nucleic acid molecules, a sequenceor structure of a particular nucleic acid molecule may be describedherein according to the normal convention of providing the sequence inthe 5′ to 3′ direction. In some embodiments of the invention, nucleicacids or polynucleotides are “isolated.” This term, when applied to anucleic acid molecule, refers to a nucleic acid molecule that isseparated from sequences with which it is immediately contiguous in thenaturally occurring genome of the organism in which it originated. Forexample, an “isolated nucleic acid” may comprise a DNA molecule insertedinto a vector, such as a plasmid or virus vector, or integrated into thegenomic DNA of a prokaryotic or eukaryotic cell or non-human hostorganism. When applied to RNA, the term “isolated polynucleotide” refersprimarily to an RNA molecule encoded by an isolated DNA molecule asdefined above. Alternatively, the term may refer to an RNA molecule thathas been purified/separated from other nucleic acids with which it wouldbe associated in its natural state (i.e., in cells or tissues). Anisolated polynucleotide (either DNA or RNA) may further represent amolecule produced directly by biological or synthetic means andseparated from other components present during its production.

For recombinant production of an antibody according to the invention, arecombinant polynucleotide encoding it may be prepared (using standardmolecular biology techniques) and inserted into a replicable vector forexpression in a chosen host cell, or a cell-free expression system.Suitable host cells may be prokaryote, yeast, or higher eukaryote cells,specifically mammalian cells. Examples of useful mammalian host celllines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL1651); human embryonic kidney line (293 or 293 cells subcloned forgrowth in suspension culture, Graham et al., J. Gen. Virol. 36:59(1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamsterovary cells/−DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod.23:243-251 (1980)); mouse myeloma cells SP2/0-AG14 (ATCC CRL 1581; ATCCCRL 8287) or NS0 (HPA culture collections no. 85110503); monkey kidneycells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76,ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2);canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human livercells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51);TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC5 cells; FS4 cells; and a human hepatoma line (Hep G2), as well as DSM'sPERC-6 cell line. Expression vectors suitable for use in each of thesehost cells are also generally known in the art.

It should be noted that the term “host cell” generally refers to acultured cell line. Whole human beings into which an expression vectorencoding an antigen binding polypeptide according to the invention hasbeen introduced are explicitly excluded from the definition of a “hostcell”.

Antibody Production

In an important aspect, the invention also provides a method ofproducing antibodies of the invention which comprises culturing a hostcell (or cell free expression system) containing polynucleotide (e.g. anexpression vector) encoding the antibody under conditions which permitexpression of the antibody, and recovering the expressed antibody. Thisrecombinant expression process can be used for large scale production ofantibodies, including IL-22R antibodies according to the invention,including monoclonal antibodies intended for human therapeutic use.Suitable vectors, cell lines and production processes for large scalemanufacture of recombinant antibodies suitable for in vivo therapeuticuse are generally available in the art and will be well known to theskilled person.

Therapeutic Utility of IL-22R Antibodies

The IL-22R antibodies provided herein can be used as medicaments,particularly for use in the treatment or prophylaxis of disorders wherethe pathology is attributable to dysregulated signalling via the cellsurface IL-22R complexes. Such dysregulated signalling may be linked tooverexpression or overproduction of any one of the cytokines IL-22,IL-20 and/or IL-24.

The term “treating” or “treatment” means slowing, interrupting,arresting, controlling, stopping, reducing severity of a symptom,disorder, condition or disease, but does not necessarily involve a totalelimination of all disease-related symptoms, conditions or disorders.The term “prophylaxis” means preventing the onset of a disorder,condition or disease or preventing the onset of symptoms associated witha disorder, condition or disease.

The ligands that signal via the IL-22R have been implicated in a numberof diseases and since IL-22R is selectively expressed on skin andepithelial cells, the key diseases are those affecting skin andepithelia including but not limited to psoriasis, psoriatic arthritisand atopic dermatitis. High levels of IL-22 have been found in humanpsoriatic plaques (Boniface et al., Clin Exp Immunol. 150: 407-415(2007)) and the involvement of this cytokine in the pathogenesis ofpsoriasis has been demonstrated experimentally in mouse models of skininflammation (Ma et al., J Clin Invest. 118: 597-607 (2008); Van Belleet al. J Immunol. January 1; 188(1):462-9 (2012)).

In certain embodiments, provided herein are methods of treating skininflammatory diseases. In certain embodiments, provided herein aremethods of treating skin inflammatory diseases selected from psoriasis,psoriatic arthritis, contact dermatitis or atopic dermatitis in a humansubject. The methods comprise administering to a patient in need thereofa therapeutically effective amount of any of the IL-22R antibodies orantigen binding fragments as defined elsewhere herein. All embodimentsof the IL-22R antibodies of antigen binding fragments as describedherein are equally applicable to the methods of treatment of the presentinvention.

In certain embodiments, provided herein are methods of treating Sjögrensyndrome, or cancers selected from hepatocarcinoma, liposarcoma, oralsquamous cell carcinoma, colon and colorectal cancer, pancreatic cancer,small- and large-cell lung cancer, breast cancer, glioblastoma,cutaneous T-cell lymphoma, anaplastic large cell lymphoma, mantle celllymphoma, in a human subject which methods comprise administering to apatient in need thereof a therapeutically effective amount of any of theIL-22R antibodies or antigen binding fragments as defined elsewhereherein. All embodiments of the IL-22R antibodies of antigen bindingfragments as described herein are equally applicable to the methods oftreatment of the present invention.

For human therapeutic use the IL-22R antibodies described herein may beadministered to a human subject in need of treatment in an “effectiveamount”. The term “effective amount” refers to the amount or dose of aIL-22R antibody which, upon single or multiple dose administration to ahuman patient, provides therapeutic efficacy in the treatment ofdisease. Therapeutically effective amounts of the IL-22R antibody cancomprise an amount in the range of from about 0.1 mg/kg to about 20mg/kg per single dose. The amount of antibody administered at any giventime point may be varied so that optimal amounts of IL-22R antibody,whether employed alone or in combination with any other therapeuticagent, are administered during the course of treatment.

It is also contemplated to administer the IL-22R antibodies describedherein, or pharmaceutical compositions comprising such antibodies, incombination with any other suitable treatment for the diseasesidentified above, as a combination therapy.

Pharmaceutical Compositions

The scope of the invention includes pharmaceutical compositions,containing one or a combination of IL-22R antibodies of the invention,or antigen-binding fragments thereof, formulated with one or more apharmaceutically acceptable carriers or excipients. Such compositionsmay include one or a combination of (e.g., two or more different) IL-22Rantibodies. Techniques for formulating monoclonal antibodies for humantherapeutic use are well known in the art and are reviewed, for example,in Wang et al., Journal of Pharmaceutical Sciences, Vol. 96, pp 1-26,2007, the contents of which are incorporated herein in their entirety.

In certain embodiments, the pharmaceutical compositions are formulatedfor administration to a subject via any suitable route of administrationincluding but not limited to intramuscular, intravenous, intradermal,intraperitoneal injection, subcutaneous, epidural, nasal, oral, rectal,topical, inhalational, buccal (e.g., sublingual), and transdermaladministration. In preferred embodiments, the composition is formulatedfor subcutaneous administration.

INCORPORATION BY REFERENCE

Various publications are cited in the foregoing description andthroughout the following examples, each of which is incorporated byreference herein in its entirety.

EXAMPLES

The invention will be further understood with reference to the followingnon-limiting examples.

Example 1 Immunization of Llama

Two llamas were immunized with a mixture of recombinant human IL22Rprotein (R&D systems) and human Fn14-Fc according to the immunizationschedule shown in Table 3 below. After six weekly injections ofrecombinant proteins, blood was collected and the sera of the immunizedllamas were used to measure the humoral immune response against IL22R bydetecting the presence of antibodies against the immunized antigenbefore immunization and after immunization. Both llamas produced asignificant and specific immune response against IL22R.

TABLE 3 Immunization schedule for each Ilama Week Date Day AntigenTissue collection 10 ml pre-immune blood (serum) 39 Sep. 29, 2010 0IL-22R (80 μg) + — Fn14-Fc (80 μg) 40 Oct. 7, 2010 8 IL-22R (40 μg + —Fn14-Fc (40 μg) 41 Oct. 14, 2010 15 IL-22R (40 μg) + — Fn14-Fc (40 μg)42 Oct. 21, 2010 22 IL-22R (40 μg) + — Fn14-Fc (40 μg) 43 Oct. 28, 201029 IL-22R (40 μg) + — Fn14-Fc (40 μg) 44 Nov. 04, 2010 36 IL-22R (40μg) + — Fn14-Fc (40 μg) 45 Nov. 08, 2010 40 400 ml immune blood 10 mlimmune blood (plasma)

Example 2 Library Construction and Selection

After immunization, PBMCs were harvested and RNA was extracted. Randomprimed cDNA synthesis was carried out and the llama VHCH1, VλCλ and VκCκgene segments were PCR amplified. Two approaches were used to PCRamplify the light chains. The first approach used a primary PCRamplification with primers without restriction site linkers, followed byPCR amplification with primers tagged with restriction sites (ApaLI andAscI). For the second approach, the tagged primers were used to amplifythe cDNA directly. The VHCH1 libraries were built using a two-step PCR,in which 25 cycles with non-tagged primers were carried out followed by10 cycles using tagged versions of these primers (containing SfiI andNotI restriction sites) (see WO2010/001251).

The PCR-amplified light chains were digested with ApaLI and AscI whilstthe PCR-amplified heavy chains were digested with SfiI and NotI, andcombined into a Fab library by cloning the light chain library insertinto heavy chain library pCB3 vector using ApaLI and AscI restrictionsites. The final Fab libraries were found to be of requisite diversity>10⁹ different clones).

After library construction, phages were produced and phage displayselection was performed on human IL22R (Biotechne, 2770-LR). Forselection of IL22R-specific clones, human IL22R was (i) coated directlyto a MaxiSorp™ plate (Nunc); or (ii) captured with a non-competitiveanti-human IL22R antibody (MAB2770, R&D systems); or (iii) captured withneutravidin after biotinylation. The coating and capture of IL22R wasusually done in two different concentrations; for example 5 μg/ml and0.1 μg/ml.

After the first round of selection no clear enrichment was seen butafter the second and third round, dose-dependent enrichments in alllibraries were observed; up to 100-fold after the second round and10,000-fold after the third round. The enrichments on antibody capturedIL22R were found to be higher compared to those on the directly coatedIL22R. This could be due to the fact that the IL22R is only 25 kDa andthe direct coating affects its conformation or limits the availableepitope. Therefore a third selection campaign was done usingbiotinylated hIL22R.

In addition to the selections described above, two extra rounds ofselection were performed on biotinylated recombinant mouse IL22R after afirst round of selection on MAB2770-captured human IL-22R. Thisselection was done to identify Fab cross-reactive with mouse IL22R. As apositive control on the selections, selection on neutravidin-capturedhuman IL-22R-biotin was conducted in parallel. Overall the selectionprocess was very successful with significant enrichment observed forIL22R Fab binders.

Example 3 Screening for IL22R Specific Fabs

After the successful phage display selection, the Fabs present inperiplasm were tested for their ability to block binding of IL-22 to theIL22R. In addition, screening was carried out to identify the clonebinding to the mouse IL22R1. A new approach was developed based onsurface plasmon resonance (SPR), which allowed testing for both ligandcompetition, affinity to human and mouse cross-reactivity in one screen.

The different channels of a Biacore chip (CM5) were coated with:

1. Nothing (Blank)

2. IL-22 (3000 RU) to test for competitive activity of the Fab (whenIL22R is co-injected)

3. Human IL22R (3000 RU) to test for binding to the target

4. Mouse IL22R (2500 RU) to test for cross-reactivity

To test for ligand competition, a low concentration of soluble IL22R1(0.2 μg/ml) was premixed with the periplasmic extracts before injection.In this way, it was also possible to measure the ability of the Fab toblock the ligand-receptor interaction on channel 2. The periplasmicextracts obtained were screened for binding to human IL22R (channel 3)and mouse IL22R (channel 4).

The advantage of this method compared to conventional ELISA (binding orcompetitive) is that several characteristics of the Fab are tested atthe same time: 1) binding to the target human IL-22R; 2) competitiveactivity; and 3) cross-reactivity. Furthermore, the measure of theoff-rates gives a strong indication of the affinity of the Fabs tested.

Using this alternative screening method, Fabs with all possiblecharacteristics were identified and categorised based on their off-rate:

-   -   Competing or non-competing    -   Cross-reactive and non-cross-reactive    -   Competing and cross-reactive    -   Non-competing and cross-reactive

Various clones were then sent for sequencing.

Example 4 Sequence Analysis of IL-22R Fabs

Clones identified by the Biacore analysis (e.g. ligand competition,maximal binding, low off-rate or mouse cross-reactivity) were sequenced.The clones were then grouped based on their CDR3 identity to form VHfamilies.

In total, 67 different VHs belonging to 13 different VH families wereidentified. Many more light chain families (more than 80 Vlambda andVkappa sequences) were identified. The large diversity in sequence andfunction of the Fabs show that the immunization and selection were verysuccessful.

The VH and VL domain amino acid sequences of clones of particularinterest are shown in Tables 4-6 below, and polynucleotide sequences ofthese clones are shown in Table 7.

TABLE 4Framework regions and CDR sequences for VH domains of IL-22R Fabs SEQSEQ SEQ SEQ SEQ SEQ SEQ ID ID ID ID ID ID ID Clone FR1 NO. CDR1 NO. FR2NO. CDR2 NO. FR3 NO. CDR3 NO. FR4 NO. 157A2 QVQLVE 1 SYDM 2 WVRQA 3SIYNDG 4 RFTISRDNAKN 5 VGFSGT 6 WGQG 7 SGGGLV S PGKGL SNTAYS TLYLQMNSLKSYYSES TQVT QPGGSL EWVS DSVKGS EDTAVYYCAK VSS RLSCAA DSVKG SGFTFS 166G8QVQLVE 8 SYFMS 9 WVRQA 10 GIHISG 11 RFTISRDNAKN 12 PPGPFKA 13 WGKGTL 14SGGGLV PGKGP GITYYL TLYLQMNNLKP HYNGMKY VTVSS QPGDSL EWVS DSVKGEDTAVYYCVT RLSCAA SGFTFG

TABLE 5Framework regions and CDR sequences for VL domains of IL-22R Fabs SEQSEQ SEQ SEQ SEQ SEQ SEQ ID ID ID ID ID ID ID Clone FR1 NO. CDR1 NO. FR2NO. CDR2 NO. FR3 NO. CDR3 NO. FR4 NO. 157A2 NFMLTQ 15 QGGY 16 WYQQK 17GNNNR 18 NTATLTISGAQ 19 QSGSSS 20 FGGGT 21 PSAVSV YAH PGQAP PS AEDEAEYYCANAV HLTVL SLGQTA VLVIY KITC 166G8 NFMLTQ 22 TGTS 23 WYQQL 24 KVNTR 25NTASLTISGLQ 26 ASYRLY 27 FGGGT 28 PPSVSG RDIG PGLAP SS SEDEADYYC ADYVHLTVL TLGKTV DYNY KLLIY TISC VS

TABLE 6 Variable domain sequences for IL-22R Fabs SEQ SEQ ID ID Clone VHNO. NO. 157A2 QVQLVESGGGLVQPGGSLRL 29 NFMLTQPSAVSVS 30SCAASGFTFSSYDMSWVRQA LGQTAKITCQGGY PGKGLEWVSSIYNDGSNTAY YAHWYQQKPGQAPSDSVKGRFTISRDNAKNTLY VLVIYGNNNRPSG LQMNSLKSEDTAVYYCAKVG IPERFSGSSSGNTFSGTYYSESWGQGTQVTVSS ATLTISGAQAEDE AEYYCQSGSSSAN AVFGGGTHLTVL 166G8QVQLVESGGGLVQPGDSLRL 31 NFMLTQPPSVSGT 32 SCAASGFTFGSYFMSWVRQALGKTVTISCTGTS PGKGPEWVSGIHISGGITY RDIGDYNYVSWYQ YLDSVKGRFTISRDNAKNTQLPGLAPKLLIYK LYLQMNNLKPEDTAVYYCV VNTRSSGTPDRFS TPPGPFKAHYNGMKYWGKGGSKSGNTASLTIS TLVTVSS GLQSEDEADYYCA SYRLYADYVFGGG THLTVL

TABLE 7 Polynucleotide sequences for IL-22R Fabs SEQ SEQ ID ID Clone VHNO. VL NO. 157A2 CAGGTGCAGCTCGTGGAGTC 52 AATTTTATGCTGA 73TGGGGGAGGCTTGGTGCAGC CTCAGCCCTCCGC CTGGGGGTTCTCTGAGACTC GGTGTCCGTGTCTTCCTGTGCAGCCTCTGGATT TTGGGACAGACGG CACCTTCAGTAGCTACGACA CCAAGATCACCTGTGAGCTGGGTCCGCCAGGCT CCAAGGGGGCTAT CCAGGGAAGGGGCTGGAGTG TATGCTCACTGGTGGTGTCCAGTATTTATAATG ACCAGCAGAAGCC ACGGTAGTAACACAGCCTAT AGGCCAGGCCCCTTCAGACTCCGTGAAGGGCCG GTGTTGGTCATCT ATTCACCATCTCCAGAGACA ATGGAAATAATAAACGCCAAGAACACGTTGTAT TAGGCCCTCAGGG CTGCAAATGAACAGCTTGAA ATCCCTGAGCGCTATCTGAGGACACGGCCGTGT TCTCTGGCTCCAG ATTACTGTGCAAAAGTTGGC TTCTGGGAACACATTAGTGGTACTTACTACAGT GCCACCCTGACCA GAATCATGGGGCCAGGGGAC TCAGCGGGGCCCACCAGGTCACCGTGTCCTCA GGCTGAGGACGAG GCCGAGTATTACT GTCAGTCAGGAAGCAGTAGTGCTAAT GCTGTGTTCGGCG GAGGGACCCATCT GACCGTCCTG 166G8CAGGTGCAGCTGGTGGAGTC 74 AATTTTATGCTGA 75 TGGGGGAGGCTTGGTGCAGCCTCAGCCTCCCTC CTGGGGATTCTCTGAGACTC CGTGTCTGGAACT TCCTGTGCAGCCTCTGGATTCTGGGAAAGACGG CACCTTCGGAAGCTATTTCA TCACCATCTCCTG TGAGCTGGGTCCGCCAGGCTCACTGGAACCAGT CCAGGAAAGGGGCCCGAGTG CGTGACATTGGGG GGTCTCAGGTATTCATATTAACTATAACTATGT GTGGTGGTATTACATACTAC CTCCTGGTATCAA TTAGACTCCGTGAAGGGCCGCAGCTCCCAGGAT ATTCACCATCTCCAGAGACA TGGCCCCCAAACT ACGCCAAGAACACGCTGTATCCTGATCTATAAA CTGCAAATGAACAACCTGAA GTCAACACTCGAT ACCTGAGGACACGGCCGTGTCCTCAGGGACCCC ATTATTGTGTAACACCCCCG TGATCGCTTCTCT GGCCCCTTTAAGGCCCATACGGCTCCAAGTCAG AATGGCATGAAGTACTGGGG GCAACACGGCCTC CAAAGGGACCCTGGTCACCGCCTGACCATCTCT TCTCCTCA GGGCTCCAGTCTG AGGACGAGGCTGA TTATTACTGTGCCTCATATAGACTGT ACGCCGATTATGT GTTCGGCGGAGGG ACCCATCTGACCG TCCTG

Example 5 Characterisation of Fabs

The clones with the best off-rates were re-cloned into pCB4, expressedand purified by IMAC (Talon from Clonentech). These clones were testedagain using Biacore. The results of the further Fab characterisation areshown in Table 8 below.

TABLE 8 Characterisation of IL-22R binding Fabs Binding to Binding tohuman IL- mouse IL- IL-22 VH family Clone 22R 22R blocking Off-rate(s⁻¹⁾ 1 170B2 Yes Yes* Yes 3.5E−04 1 170D6 Yes Yes* Yes 5.8E−03 1 160C8Yes No Yes 3.2E−03 2 160C2 Yes Yes No 1.7E−03 3 158C4 Yes Yes No 6.7E−034 157A2 Yes No Yes 3.7E−03 4 171F4 Yes No Yes 5.9E−03 4 171A1 Yes No Yes4.1E−03 4 171B1 Yes No Yes 1.9E−03 4 171C1 Yes No Yes 3.5E−03 5 159B8Yes No Yes 1.5E−03 5 166G8 Yes No Yes 8.8E−04 5 169C1 Yes No Yes 3.2E−045 169H7 Yes No Yes 4.7E−04 5 169C8 Yes No Yes 4.3E−04 5 169H10 Yes NoYes 6.5E−04 5 171F10 Yes No Yes 1.2E−03 6 157C8 Yes Yes Yes 4.3E−03 6157G8 Yes Yes Yes 8.6E−04 6 171A8 Yes Yes Yes 4.8E−03 7 157B8 Yes No No7.7E−04 8 158H4 Yes No No 2.9E−03 11 165B8 Yes Yes No 9.7E−04 19 169G4Yes — No 7.3E−04 20 172C9 Yes Yes No 6.1E−04 22 166D8 Yes No No 3.8E−03*observed only once with purified Fab (not peri or mAb) suggesting verylow affinity

Depending on the diversity in the VH family, competing activity andaffinity (measured using the k_(off) obtained in the Biacore) one orseveral clones from each family were selected and reformatted intomonoclonal antibodies (mAb) for further characterisation.

Example 6 Characterization of IL-22R Binding mAbs

As noted above, selective clones of each VH family were produced asIgG1. After re-cloning of the HC and LC variable regions into separatepUPE expression vectors containing the human constant domains, HEK293Ewere transiently transfected with the two plasmids, one encoding theentire heavy chain and a second encoding the light chain. Thetransfected cells were allowed to express the antibodies during a 6-daycell culture period. After antibody purification from cell culturesupernatant using protein A beads, the purified antibodies were testedfor their ability to neutralize human and mouse IL22R signalling usingdifferent cell lines as described below.

-   -   BW-hIL22R cell line: Is derived from the BW cell line and has        stable expression of human IL22R which introduces IL22-dependent        growth arrest. Therefore the cells will proliferate only when        the IL22-IL22R interaction is blocked. A potent antibody will        promote proliferation at low antibody concentration.        Proliferation is measured by thymidine incorporation.    -   Baf3-mIL22R cell line: Is derived from the Baf3 cell line and        has stable expression of mouse IL22R which gives rise to an        IL22-dependent proliferation. Antibody neutralizing the mIL22R        will block the proliferation in an antibody concentration        dependent manner.    -   Baf3-hIL22R/IL20Rb cell line: Is derived from the Baf3 cell line        but co-expresses hIL22R with hIL20Rbeta allowing the cells to        proliferate in the presence of IL20. Antibody neutralizing the        hIL22R will block the proliferation of cells stimulated with        IL20.

The results of testing various IL22R mAbs against the BW-hIL22R cellline and Baf3-hIL22R/IL20Rb cell line are shown in FIGS. 4A-4B and Table9. In FIG. 4A, the effects of IL22R neutralizing antibodies in restoringproliferation of the BW-hIL22R cell line can be seen. In FIG. 4B theeffects of IL22R neutralizing antibodies in inhibiting IL-20proliferation of the IL-22R expressing cell line can be seen. The IL22Rantibody described in WO2011/061119 (280.346.TSY) was included in theexperiments as a benchmark or reference IL-22R antibody.

In addition to the potency on cells, the in vitro binding kinetics andblocking activity of the purified antibodies were measured in Biacore(3000). For affinity measurement, recombinant IL22R was coated onto aBiacore CM5 chip at 200 RU using a standard coating protocol (IL22R isinjected at 10 μg/ml in Acetate buffer pH 4.5). Antibodies tested at arange of concentrations and prepared in HBSEP buffer at pH 7.4 (Biacore)were injected at 30 μl/min for 60 second and washed with HBSEP buffer atpH 7.4 (Biacore) for 10-20 min. The obtained sensograms were analyzedwith the BIAevaluation software and kinetics were determined usingstandard fitting (Langmuir 1:1 with mass transfer).

For blocking activity using the Biacore 3000, recombinant IL22 (RnDSystem) was coated at 2000 RU onto a CM5 Chip. The antigen bindingdomains (purified IgG, purified Fabs or periplasmic extract containingthe Fabs) were pre-incubated with recombinant human IL22R (concentrationbetween 0.2 and 1 μg) before injection into the hIL22 coated channel.Detection of binding (IL22R:IL22) indicated that the antigen bindingdomain was not competing while when no binding (IL22R:IL22) wasdetected, this indicated that the antigen binding domain was blockingthe IL22:IL22R interaction in vitro. The results of the Biacore andproliferation assays are summarized in Table 9 below.

TABLE 9 Summary of the characteristics of a panel of hIL22R antibodiesPotency in proliferation assay IC50 (pM) Binding to hIL22R in BiacoreBlocking BW BaF3 VH Kd (1/s) IL22-IL22R human mouse Family Clone Ka(1/Ms) (off-rate) KD (M) Biacore IL22R IL22R 1 170B2 7.6E+05 9.3E−051.2E−10 Yes 60-200* — 1 170D6 2.2E−04 Yes 3,830 — 2 160E2  6E−05 No — 1,406 3 158C4  6E−04 No — — 4 157A2 3.9E+05 1.1E−03 2.8E−09 Yes 80-130*— 4 171A1 7.4E−04 3,320 5 159B8  2E−03 Yes 100-200*  — 5 166G8 9.2E+062.0E−04 2.2E−11 Yes 10-50*  — 5 169C1 Yes 35-90*  (—) 6 157G8 5.4E−05Yes 1,174/2,090 — 7 157B8 3.4E−05 No 19,000  38,000 8 158H4  3E−05 No 500-1,600* 5-20 × 10³* 11 165B8 3.1E−06 No — — 19 169G4 5.4E+05 3.1E−035.7E−09 No 1,489 20 172C9 No — — 22 166D8 3.2E−03 No 74,000  — Benchmark280.346.TSY 2.7E+05 6.4E−05 2.4E−10 Yes 20-110* 10-50* *range observedover several experiments

IL22R antibodies 157A2 and 166G8 display picomolar potency.

Example 7 Species Cross-Reactivity of IL-22R mAbs

The IL22R mAbs were tested for species cross-reactivity against murineIL-22R and non-human primate IL-22R.

The cDNA for cynomolgus IL22R was not available and had to be extractedfrom cynomolgus cDNA libraries. Primers were designed based on the NCBIpublic database. Several sequences for cynoIL22R ECD were available frompublic databases (Genbank, NCBI) and contained either insertion,deletion or both (see FIGS. 5A-5B). The uncertainty regarding the truecynomolgus IL22R sequence justified the cloning from cyno cDNA.

The cynomolgus IL22R ECD was amplified by PCR from a cDNA library andcloned in frame downstream of the IgGkappa signal peptide and upstreamof the human Fc in the pUPE vector. Although the cDNA used was derivedfrom cynomolgus, sequencing of several clones showed that the rhesusIL22R was also cloned. Rhesus IL22R-ECD was characterized by the absenceof the deletion and insertion found in the cynomolgus (boxed in FIGS.5A-5B) and by 2 amino acid differences between rhesus and cyno IL22R (inbold in FIGS. 5A-5B).

After production and purification of the cyIL22R-Fc and the rhIL22R-Fc,the binding specificity to cynomolgus, rhesus and mouse IL22R ECD of allthe antibodies from primary selection was tested in ELISA and by SPR(Biacore). Monomeric mouse IL22R was purchased from R&D Systems (cat4248-MR). The results are summarized in Table 10.

TABLE 10 Species cross-reactivity of IL-22R mAbs Binding to IL22R from:VH Family Clone human mouse cyno rhesus 1 170B2 Yes No nt nt 1 170D6 YesNo nt nt 2 160E2 Yes Moderate Yes Yes 3 158C4 Yes No Yes Yes 4 157A2 YesNo Yes Yes 5 159B8 Yes No No Weak 5 166G8 Yes No No Yes 5 169C1 Yes NoNo Weak 6 157G8 Yes Moderate Yes Moderate 7 157B8 Yes No Yes Yes 8 158H4Yes Moderate Yes Yes 8 205A5 Yes Moderate Yes Yes 11  165B8 Yes ModerateModer- Moderate ate 19  169G4 Yes No Yes Yes 20  172C9 Yes Moderate YesYes 22  166D8 Yes No Yes Yes Benchmark 280.346.TSY Yes Yes Yes Yes nt =not tested

Example 8 Epitope Mapping

Two methods were employed to identify and compare the epitopes bound bythe IL22R mAbs: competitive ELISA and FACS.

8.1 Epitope Mapping Using Competitive ELISA

The epitopes recognized by the antibodies were compared to each other bycompetitive ELISA. At least one mAb representative of each VH family wascoated in a Maxisorp plate. Subsequently, biotinylated human IL22R wasadded in the presence of a large excess of the mAbs to test. The bindingof biotinylated IL22R to the coated antibody was detected usingHRP-conjugated streptavidin. When biotinylated IL22R was not detected,meaning that the soluble mAb prevented binding to the coated mAb, it wasdetermined that both mAbs bind to the same epitope, or have anoverlapping epitope. The epitope mapping was further refined by usingFabs instead of the mAbs. The smaller Fab fragment allowed for somedistinction of very close epitopes.

A variety of epitopes were identified for antibodies of VH families 1-8,10, 11, 19 and 22. The epitope mapping shows a very broad epitopecoverage for such a small protein (25 kDa). As shown in FIG. 6, theepitopes were grouped according to whether the antibodies (i) blockedIL-22 binding in vitro and neutralised IL-22 signalling in a cell-basedassay (bottom left quadrant); (ii) blocked IL-22 binding in vitro buthad no neutralising activity (top right quandrant); or (iii) did notblock IL-22 binding in vitro but did have neutralising activity in acell-based assay (bottom right quadrant). Six overlapping butdistinguishable epitopes were found to be blocking and neutralising (seebottom left quadrant) and out of these six epitopes, two (mAbs fromfamily 4 and 22) had an overlapping epitope with the benchmark antibody(280.346.TSY, see patent application WO2011/061119). The epitope was notidentical to the benchmark since these mAbs (from family 4 and 22) wereable to compete also with all the mAbs from families 1, 5, 6 and 7 eventhough these were not able to compete the Benchmark antibody. Takentogether these data suggest 2 distinct epitope groups, one formed by theBenchmark and one formed by mAbs from families 1, 5, 6 and 7, and athird group (mAbs from fam 4 and 22) which overlap with both groups(FIG. 6).

As shown in the top right-hand quadrant of FIG. 6, 3 antibodies wereidentified that bind to IL22R but do not block IL22 binding orneutralize activity in vivo. These antibodies likely bind regions ofIL22R not involved in ligand binding or activation of signaling andcould be used for pure detection. The 3 antibodies shown in the bottomright-hand quadrant of FIG. 6 are unusual in that they neutralize IL22Ractivity in vivo but do not block IL22 in vitro, suggesting a newfunctional epitope and unexpected mode of action.

8.2 Epitope Mapping Using FACS Analysis

Jones et al. (Structure 16(9): 1333-1344 (2008)) reports the structureof IL-22 bound to the extracellular domain of IL22R1. In this paper, theauthors show that the two IL22R domains, called D1 and D2, interact withthe IL-22 ligand at site 1A and site 1B, respectively (see FIGS. 7A-7B7). Two key residues in IL22R-D1 involved in the direct interaction withthe site 1A of IL22 are lysine 58 (K58) and tyrosine 60 (Y60).Tryptophan 208 (W208) of IL22R-D2 is directly involved in theinteraction with the site 1B of IL22 (see FIGS. 7A-7B).

The binding of IL22R antibodies and of the benchmark antibody(280.346.TSY) to BW cells over-expressing various IL22R mutants wastested. The IL22R mutants have mutations of amino acids known to beinvolved in the IL-22/IL22R interaction. In this setup, the antibodieswere biotinylated, added to the cells and binding of the antibody toIL22R was detected with labeled streptavidin by Flow cytometry (FACS).Table 11 summarizes the results obtained.

TABLE 11 Antibody binding to cells expressing IL22R mutants hIL-22Rmutants A209D W208A T207A K58A Y60A R112A T89A E90A Q117A D162A IL22binding site VH Fam 1B 1B 1B 1A 1A 1A 1A 1A 1A/B 1B AMR22 + + + X XX + + + + 280.346.TSY + + + X X + + + + + 198A1 5 X + + + + + + + + +166G8 5 + + + + + + + + + + 197B7 1 + + + + + + + + + + 196F81 + + + + + + + + + + 158H4 8 + + + + + + + + + + 169G419 + + + + + + + + + + + = binding; X = mutation in IL22R results inloss of antibody binding.

The FACs results clearly show that the benchmark antibody 280.346.TSYinteracts with residues of the D1 domain (K58 and Y60). The same is truefor a second control IL-22R antibody, AMR22, although the epitope is notcompletely the same, since the R112A mutation affects AMR22 binding. Asexpected from the ELISA epitope mapping showing that certain llamaantibodies do not compete with 280.346.TSY, the other antibodies testedwere not affected by the mutations affecting the binding of 280.346.TSYto IL22R. This confirmed that the antibodies target another epitope,possibly including residues in the D2 domain. Indeed, the A209D mutationin the D2 domain of IL22R abrogated binding of 198A1 confirming that198A1 and the antibody from family 5 bind to the D2 domain of IL22R (seeFIG. 7B).

Surprisingly antibody 166G8, which shares exactly the same VH as 198A1,but has a different VL, is not affected by the A209D mutation suggestingthat the VL is involved in the interaction. Presumably, the VL of 166G8allows for a large residue at position 209, while the VL of 198A1 doesnot. This hypothesis is confirmed by their binding specificity to rhesusIL22R. In rhesus, IL22R has a serine at position 209. Since 166G8 allowsfor a large amino acid at that position, it can bind rhesus IL22R while198A1 is not able to bind rhIL22R since it does not allow largeresidues.

Example 9 Neutralization of IL20 and IL24 Signalling in a Cell-BasedAssay

IL22R antibodies were tested for their ability to block IL-20 and IL-24dependent signaling in cells co-expressing IL22R and IL20Rb. Amechanistic difference exists between the IL-22, IL-20 and IL-24dependent receptor activation. It is believed that IL-22 inducessignaling by first binding to the IL22R, triggering the co-receptorIL10R2 recruitment and activation of the trimeric complex. For IL-20, itis believed that this cytokine binds the co-receptor first (IL20R2)before recruiting IL22R and activation of the trimeric complex. Finallyfor IL-24, it is not clear how the receptor complex is recruited andactivated, nor is it clear where IL-24 binds.

9.1 IL-24 Signalling

Several cell lines were used to test the IL-24 dependent signaling,Baf3-hIL22R/20R2 or Baf3-mIL22R/20R2. IL-24 induced proliferation ofthese cell lines with a significant window as measured by thehexosaminidase level (read-out used to count cells) ranging from OD 0.17to 1.5 for the Baf3-hIL22R/20Rb cells and ranging from 0.15 to 0.8 forthe Baf3-mIL22R/20Rb cells. None of the antibodies tested had an effecton the IL-24 induced proliferation. These results suggest that eitherIL-24 has a completely different mode of IL22R activation by binding toa site very distinct to IL22 and IL-20, or that IL22R is not involved inthe IL-24 signaling in the cell line tested (and the assay is notpredictive of neutralizing activity).

9.2 IL-20 Signalling

Baf3-hIL22R/IL20Rb cell line is derived from the Baf3 cell line butco-expresses hIL22R with hIL20Rβ allowing the cells to proliferate inthe presence of IL-20. Antibody neutralizing the hIL22R will block theproliferation of cell stimulated with IL-20. Results produced testingllama antibodies alongside the 280.346.TSY and AMR22 benchmarkantibodies are shown in FIG. 8.

The IL-20 proliferation on Baf3-hIL22R/IL20Rb was tested in parallelwith the IL-22 proliferation assay of BW-hIL22R, allowing a goodcomparison between the two cytokines. The results of up to threeindependent experiments are shown in Table 12.

TABLE 12 Potency of the antibodies in blocking IL-22 and IL-20 signalingvia IL22R IC50 blocking IC50 blocking VH family IL-22 (pM) IL-20 (pM)166G8 5  6-84 160-170 198A1 5cs 10-80  85-140 197B7 1  61-3701,600-6,960 157A2 4 131-540 184-545 218A7 4  360 nt 158H4 8 1600 nc205A5 8cs 500-1,200 nc AMR22 Benchmark   715-5,000 470 (n = 1)280.346.TSY Benchmark  22-250 280-800 *Range observed over up to threeexperiments (2250, 2292, 2301 for IL-20 and 2253, 2292, 2301, 2428 forIL-22); nc: not competing; nt: not tested; cs: chain shuffled

It can be concluded that all the antibodies from families #1, #4 and #5were able to block both IL-20 and IL-22. It is difficult to compare thepotency between the IL-22 and IL-20-stimulated proliferation because ofthe difference in sensitivity, which depends on the cytokineconcentration, receptor number and intracellular biological variation.However, most antibodies were found to be more potent in blocking IL-22signaling compared to blocking that of IL-20. The interesting exceptionwas 157A2, which has the same potency to block IL-22 and IL-20.

Example 10 Selection of Lead Antibodies and Germlining

The affinity of the most interesting antibodies (neutralizing IL22signaling) was further improved by light chain shuffling. During lightchain shuffling the VHCH1 of the selected antibody is combined with thelibrary of light chain from the same llama from which the VHCH wasfound. Several rounds of off-rate phage display selection are thenperformed to identify the VHCH:VLCL pairs which have the best affinity.In the case of antibody 166G8, several clones with improved bindingkinetics were found including 198A1, but unfortunately, this clone lostmost of its rhesus cross-reactivity and therefore 166G8 remained themost attractive candidate of family #5 for development. In the case of157A2 (from family #4), several Fab (e.g. 218A7) were identified withimproved affinity as compared to 157A2. The following antibodies weretaken forward for further development. These antibodies were selectedbased on potency, species cross-reactivity and epitope or mode ofaction.

-   -   218A7 (an improved variant of 157A2, family #4)—this antibody        was found to have very good potency against IL-22 and IL-20 (sub        nM), is cross-reactive with non-human primate IL22R but is not        cross-reactive with the murine IL22R. 157A2 was found to have an        epitope on IL22R distinct but overlapping with the 280.346.TSY        benchmark antibody.    -   166G8 (family #5)—this antibody was found to have high potency        (low pM) and has some species cross reactivity, since it binds        to the rhesus IL22R (but not cynomolgus, nor murine IL22R).

The characteristics of the IL22R antibodies taken forward are summarizedin Table 13 below.

TABLE 13 Characteristics of the Ilama IL22R antibodies selected forgermlining Potency Relative VH Cross-reactivity IL-22 IL-20 potency IL-Epitope Family h cyno rh m (pM) (pM) 22 v IL-20 IL22R 157A2 4 +++ ++++++ − 131-570 184-545 1.09 D1/D2 218A7 4 +++ +++ +++ − 350-360 nd —D1/D2 166G8 5 +++ − ++ −  6-84 160-170 3.67 D2 280.346.TSY +++ +++ ++++++  22-250 280-800 7.5  D1 h = human; cyno = cynomolgusl; rh = rhesus;m = mouse

Example 11 Germlining of IL-22R Antibodies 218A7 and 166G8

Germlining was carried out as described in International patentapplication no. WO2011/080350, the contents of which are incorporatedherein in their entirety. The CDRs were mutated to remove potentialpost-translational modifications together with certain other positions,which found to vary in antibodies from the same VH and VL families withidentical paring. The VH and VL domains of the germlined antibodiesproduced from llama antibodies 218A7 and 166G8 were assessed for theiridentity with human sequences. A comparison was also made with thebenchmark antibody 280.346.TSY. The results are shown in Table 14 below.

TABLE 14 Human identity of germlined IL-22R antibodies produced from218A7 and 166G8 Off-rate for Off-rate for Name of the % identity %identity % identity human IL- rhesus IL- clone for VH for VL for VH + VL22R1* 22R1* 166G8 88.5 84.8 86.7  1.22E−0.3 2.22E−02  22 224C7 93.1 93.793.4 4.19E−04 2.20E−02  6 224C6 94.3 93.7 94.0 4.34E−04 1.85E−02  21224A6 93.1 93.7 93.4 4.41E−04 4.68E−0.3 14 224C4 92.0 94.9 93.5 4.50E−044.51E−0.3 16 224G10 92.0 94.9 93.5 4.71E−04 3.75E−0.3 15 224E8 92.0 94.993.5 4.81E−04 4.51E−0.3 1 223G5 93.1 97.4 95.3 5.29E−04 5.84E−0.3 20223D3 93.1 93.7 93.4 5.73E−04 2.01E−0.3 2 223C4 94.3 96.2 95.3 5.74E−046.27E−0.3 11 223A6 90.8 96.2 93.5 5.79E−04 2.58E−0.3 13 223G1 92.0 94.993.5 5.96E−04 5.05E−0.3 8 226B9 95.4 92.4 93.9 7.14E−04 4.65E−0.3 3225A5 93.1 96.2 94.7  1.14E−0.3 1.55E−02  5 227C5 93.1 94.9 94.0 1.25E−0.3 4.98E−0.3 17 227G10 92.0 94.9 93.5  1.25E−0.3 2.73E−0.3 19225G7 92.0 94.9 93.5  1.29E−0.3 4.27E−0.3 18 225E5 92.0 94.9 93.5 1.35E−0.3 1.67E−0.3 12 225A4 90.8 96.2 93.5  1.42E−0.3 4.21E−0.3 9225A10 95.4 92.4 93.9  1.55E−0.3 1.07E−02  7 225D8 94.3 93.7 94.0 1.64E−0.3 5.11E−0.3 23 225G4 93.1 93.7 93.4  1.74E−0.3 3.75E−0.3 10225C6 95.3 92.4 93.9  1.76E−0.3 2.63E−0.3 4 225B2 94.3 94.9 94.6 2.78E−0.3 4.55E−0.3 218A7 95.4 91.1 95.4 8.10E−04 9.70E−04  13 230C9100 96.2 98.1 3.52E−04 4.36E−04  10 230B7 98.9 97.5 98.2 3.77E−048.17E−04  19 229B4 98.9 96.2 97.6 3.81E−04 4.79E−04  1 230H2 100 98.799.4 4.01E−04 1.08E−0.3 18 228F9 97.7 97.5 97.6 4.01E−04 4.34E−04  14228C9 100 96.2 98.1 4.08E−04 4.72E−04  12 232G6 100 96.2 98.1 4.14E−041.27E−0.3 11 232F5 100 96.2 98.1 4.24E−04 1.27E−0.3 5 231C11 98.9 97.598.2 4.30E−04 1.32E−0.3 6 231E12 98.9 97.5 98.2 4.35E−04 1.33E−0.3 3228F3 97.7 98.7 98.2 4.39E−04 1.25E−0.3 2 230D5 98.9 98.7 98.8 4.45E−041.32E−0.3 4 231B11 98.9 97.5 98.2 4.55E−04 1.32E−0.3 8 229E6 98.9 97.598.2 4.59E−04 5.41E−04  20 229D9 100 94.9 97.5 4.69E−04 6.84E−04  16229G1 97.7 97.5 97.6 4.77E−04 5.08E−0.3 7 231F12 98.9 97.5 98.2 5.30E−041.35E−0.3 17 228A4 97.7 97.5 97.6 5.95E−04 1.97E−0.3 15 231A8 97.7 97.597.6  1.48E−0.3 2.85E−0.3 9 229G2 98.9 97.5 98.2  1.86E−0.3 3.30E−0.3280.346.TSY 88.5% 100% 94.3% — — *Determined for Fab fragments

Antibody 230C9 was selected as a germlined variant of antibody 218A7,and antibody 223G5 was selected as a germlined variant of antibody166G8. The VH and VL domain sequences of antibodies 218A7, 230C9 and223G5 are shown in Tables 15-19 below. (The sequences of 166G8 are shownin Tables 4-7 above.)

TABLE 15Framework regions and CDR sequences for VH domains of 218A7, 230C9 and 223G5SEQ SEQ SEQ SEQ SEQ SEQ SEQ ID ID ID ID ID ID ID Clone FR1 NO. CDR1 NO.FR2 NO. CDR2 NO. FR3 NO. CDR3 NO. FR4 NO. 218A7 QVQLVE  1 SYDMS  2 WVRQA 3 SIYNDG  4 RFTISRDNAKN  5 VGFSGT  6 WGQG  7 SGGGLV PGKGL SNTAYTLYLQMNSLKS YYSES TQVTV QPGGSL EWVS SDSVKG EDTAVYYCAK SS RLSCAA SGFTFS230C9 QVQLVE 33 SYDMN 34 WVRQA 35 SIYNDA 36 RFTISRDNSKN 37 VGFSGT  6WGQG 38 SGGGLV PGKGL SNTAY TLYLQMNSLRA YYSES TLVTVS QPGGSL EWVS SDSVKGEDTAVYYCAK S RLSCAA SGFTFS 223G5 QVQLVE 39 SYFMS  9 WVRQA 40 GIHISG 41RFTISRDNAKN 42 PPGPFK 43 WGKGT 44 SGGGLV PGKGP GITYYT TLYLQMNSLRA AHYNGALVTVSS QPGGSL EWVS DSVKG EDTAVYYCVT KY RLSCAA SGFTFS

TABLE 16Framework regions and CDR sequences for VL domains of 218A7, 230C9 and 223G5SEQ SEQ SEQ SEQ SEQ SEQ SEQ ID ID ID ID ID ID ID Clone FR1 NO. CDR1 NO.FR2 NO. CDR2 NO. FR3 NO. CDR3 NO. FR4 NO. 218A7 LPVLTQP 45 QGGY 16 WYQQK46 GQNNR 47 NTATLTISGAQ 48 QSGSSS 20 FGGGT 49 SAVSVSL YAH PGQAP PSAEDEAEYYC ANAV KLTVL GQTARITC VLVIY 230C9 SYELTQ 50 QGGY 16 WYQQK 51GQNNR 47 NTATLTISRAQ 53 QSGSSS 54 FGGGT 55 PSSVSV YAH PGQAP PS AEDEADYYCSNAV KLTVL ALGQTA VLVIY RITC 223G5 QSALTQ 56 TGTS 57 WYQQL 58 EVNKR 59NTASLTISGLQ 60 ASYRLY 27 FGGGT 61 PPSVSG SDIGS PGKAP SS AEDEADYYC ADYVQLTVL SPGQSV YNYVS KLLIY TISC

TABLE 17 Variable domain sequences for 218A7,230C9, 223G5 SEQ SEQ ID IDClone VH NO. VL NO. 218A7 QVQLVESGGGLVQPGGSLRL 29 LPVLTQPSAVSVS 62SCAASGFTFSSYDMSWVRQA LGQTARITCQGGY PGKGLEWVSSIYNDGSNTAY YAHWYQQKPGQAPSDSVKGRFTISRDNAKNTLY VLVIYGQNNRPSG LQMNSLKSEDTAVYYCAKVG IPERFSGSGAGNTFSGTYYSESWGQGTLVTVSS ATLTISRAQAEDE ADYYCQSGSSSSN AVFGGGTKLTVL 230C9QVQLVESGGGLVQPGGSLRL 63 SYELTQPSSVSVA 64 SCAASGFTFSSYDMNWVRQALGQTARITCQGGY PGKGLEWVSSIYNDASNTAY YAHWYQQKPGQAP SDSVKGRFTISRDNSKNTLYVLVIYGQNNRPSG LQMNSLRAEDTAVYYCAKVG IPERFSGSGAGNT FSGTYYSESWGQGTLVTVSSATLTISRAQAEDE ADYYCQSGSSSSN AVFGGGTKLTVL 223G5 QVQLVESGGGLVQPGGSLRL 65QSALTQPPSVSGS 66 SCAASGFTFSSYFMSWVRQA PGQSVTISCTGTS PGKGPEWVSGIHISGGITYYSDIGSYNYVSWYQ TDSVKGRFTISRDNAKNTLY QLPGKAPKLLIYE LQMNSLRAEDTAVYYCVTPPVNKRSSGVPDRFS GPFKAHYNGAKYWGKGTLVT GSKSGNTASLTIS VSS GLQAEDEADYYCASYRLYADYVFGGG TQLTVL

TABLE 18 Heavy chain and light chain sequences for 230C9 and 223G5 SEQSEQ ID ID Clone Heavy chain NO. Light chain NO. 230C9QVQLVESGGGLVQPGGSLR 67 SYELTQPSSVSVALGQ 68 LSCAASGFTFSSYDMNWVRTARITCQGGYYAHWYQ QAPGKGLEWVSSIYNDASN QKPGQAPVLVIYGQNNTAYSDSVKGRFTISRDNSK RPSGIPERFSGSGAGN NTLYLQMNSLRAEDTAVYYTATLTISRAQAEDEAD CAKVGFSGTYYSESWGQGT YYCQSGSSSSNAVFGGLVTVSSASTKGPSVFPLAP GTKLTVLGQPKAAPSV SSKSTSGGTAALGCLVKDYTLFPPSSEELQANKAT FPEPVTVSWNSGALTSGVH LVCLISDFYPGAVTVATFPAVLQSSGLYSLSSVVT WKADSSPVKAGVETTT VPSSSLGTQTYICNVNHKPPSKQSNNKYAASSYLS SNTKVDKKVEPKSCDKTHT LTPEQWKSHRSYSCQVCPPCPAPELLGGPSVFLFP THEGSTVEKTVAPTEC PKPKDTLMISRTPEVTCVV SVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYQSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSRDEL TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 223G5 QVQLVESGGGLVQPGGSLR 69 QSALTQPPSVSGSPGQ 70LSCAASGFTFSSYFMSWVR SVTISCTGTSSDIGSY QAPGKGPEWVSGIHISGGINYVSWYQQLPGKAPKL TYYTDSVKGRFTISRDNAK LIYEVNKRSSGVPDRFNTLYLQMNSLRAEDTAVYY SGSKSGNTASLTISGL CVTPPGPFKAHYNGAKYWGQAEDEADYYCASYRLY KGTLVTVSSASTKGPSVFP ADYVFGGGTQLTVLGQLAPSSKSTSGGTAALGCLV PKAAPSVTLFPPSSEE KDYFPEPVTVSWNSGALTSLQANKATLVCLISDFY GVHTFPAVLQSSGLYSLSS PGAVTVAWKADSSPVKVVTVPSSSLGTQTYICNVN AGVETTTPSKQSNNKY HKPSNTKVDKKVEPKSCDKAASSYLSLTPEQWKSH THTCPPCPAPELLGGPSVF RSYSCQVTHEGSTVEKLFPPKPKDTLMISRTPEVT TVAPTECS CVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK The shaded portions of the sequencerepresent the constant regions of the heavy and light chains. The″Q″ residue shown in bold in the constant region of the heavy chainrepresents a mutation from ″N″.

TABLE 19 Polynucleotide sequences encoding the variabledomain sequences/heavy chain and light chainsequences for 218A7, 230C9 and 223G5 SEQ SEQ ID ID Clone VH domain NO.VL domain NO. 218A7 CAGGTGCAGCTCGTGGAGT 76 CTGCCTGTGCTGACT 77CTGGGGGAGGCTTGGTGCA CAGCCCTCCGCGGTG GCCTGGGGGTTCTCTGAGA TCCGTGTCTTTGGGACTCTCCTGTGCAGCCTCTG CAGACGGCCAGGATC GATTCACCTGTGCTGGTCA ACCTGCCAAGGGGGCTCTATGGACAGAATAATAG TATTATGCTCACTGG GCCCTCGGGAAGGGGCTGG TACCAGCAGAAGCCAAGTGGGTGTCCAGTATTTA GGCCAGGCCCCTGTG TAATGACGGTAGTAACACA CTGGTCATCTATGGAGCCTATTCAGACTCCGTGA CAGAATAATAGGCCC AGGGCCGATTCACCATCTC TCAGGGATCCCTGAGCAGAGACAACGCCAAGAAC CGCTTCTCTGGCTCC ACGTTGTATCTGCAAATGA GGCGCTGGGAACACAACAGCTTGAAATCTGAGGA GCCACCCTGACCATC CACGGCCGTGTATTACTGT AGCGGGGCCCAGGCTGCAAAAGTTGGCTTTAGTG GAGGACGAGGCTGAG GTACTTACTACAGTGAATC TATTACTGTCAGTCAATGGGGCCAGGGGACCCAG GGAAGCAGTAGTGCT GTCACCGTGTCCTCA AATGCTGTGTTCGGCGGAGGGACCAAGCTG ACCGTCCTC 230C9 CAGGTGCAGCTCGTGGAGT 78 TCCTACGAACTGACT79 CTGGGGGAGGCTTGGTGCA CAGCCCTCCTCGGTG GCCTGGGGGTTCTCTGAGATCCGTGGCGTTGGGA CTCTCCTGTGCTGCCTCTG CAGACGGCCAGGATC GATTCACCTTCAGTAGCTAACCTGCCAAGGAGGC CGACATGAACTGGGTCCGC TATTATGCACACTGG CAGGCTCCAGGGAAGGGGCTACCAGCAGAAGCCA TGGAGTGGGTGTCCAGCAT GGCCAGGCCCCTGTG TTATAACGACGCCAGTAACCTGGTCATCTATGGA ACAGCCTATTCAGACTCCG CAGAATAATAGGCCC TGAAGGGCCGATTCACCATTCAGGGATCCCTGAG CTCCAGAGACAACTCAAAG CGCTTCTCTGGCTCC AACACGTTGTATCTGCAAAGGCGCTGGGAACACA TGAACAGCCTGAGAGCTGA GCCACCCTGACAATC GGACACGGCCGTGTATTACAGCCGCGCCCAGGCT TGTGCGAAAGTTGGCTTTA GAGGACGAGGCTGAC GTGGTACTTACTACAGTGATATTACTGTCAGTCA ATCATGGGGCCAGGGGACC GGAAGCAGTAGTTCT CTCGTCACTGTCTCCTCAAATGCTGTGTTCGGC GGAGGGACCAAGCTG ACCGTCCTC 223G5 CAGGTGCAGCTCGTGGAGT 80CAGTCTGCCCTGACT 81 CTGGGGGCGGCTTGGTGCA CAGCCTCCCTCCGTGGCCTGGGGATTCTCTGAGA TCTGGATCTCCTGGA CTCTCCTGTGCAGCCTCTG CAGTCTGTCACCATCGATTCACCTTCAGTAGCTA TCCTGCACTGGAACC TTTCATGAGCTGGGTCCGC AGTAGTGACATTGGGCAGGCTCCAGGAAAGGGGC TCCTATAACTATGTC CCGAGTGGGTCTCAGGTAT TCCTGGTATCAACAGTCATATTAGTGGTGGTATT CTCCCAGGAAAGGCC ACATACTACACGGACTCCG CCCAAACTCCTGATCTGAAGGGCCGATTCACCAT TATGAGGTCAACAAG CTCCAGAGACAACGCAAAG CGATCCTCAGGGGTCAACACGCTGTATCTGCAAA CCTGATCGCTTCTCT TGAACAGCCTGAGAGCTGA GGCTCCAAGTCAGGCGGACACGGCCGTGTATTAT AACACGGCCTCCCTG TGTGTAACACCCCCGGGCC ACCATCTCTGGGCTCCCTTTAAGGCCCATTACAA CAGGCTGAGGACGAG TGGCGCGAAGTACTGGGGC GCTGATTATTACTGTAAAGGGACCCTGGTCACTG GCCTCATATAGACTG TCTCCTCA TACGCCGATTATGTGTTCGGCGGAGGGACC CAACTGACCGTCCTC

Example 12 Characterisation of the Germlined Antibodies 230C9 and 223G5

12.1 Binding Affinity of Germlined mAbs

The affinity of the 230C9 and 223G5 antibodies was measured using SPR(Biacore 300). In brief, 250 RU of human IL22R (Biotechne, 2770-LR) wascoated onto a CM5 chip using standard methods (EDC coupling of IL22R ata concentration of 2 μg/ml in acetate buffer pH4.5). Then variousconcentrations of antibody were injected in HBSEP+ buffer (pH 7.4) for 2minutes. The binding was monitored also during a 10 min washing(HBSEP+).

The affinities calculated using the BIAevaluation software using aLangmuir 1:1 fitting are shown in Table 20 below.

TABLE 20 Binding affinity of germlined antibodies ka (1/Ms) kd (1/s) KD(M) 223G5 6.6E+06 1.0E−04 1.6E−11 224C4 9.3E+06 9.8E−05 1.1E−11 230C96.8E+05 8.9E−05 1.3E−10 280.346.TSY 4.9E+05 3.4E−04 6.9E−10

The antibodies 223G5 and 224C4 (both germlined variants of 166G8) showedvery high affinity for IL-22R (10-20 pM) whilst antibody 230C9 had anaffinity of 0.13 nM. All of the germlined antibodies displayed higheraffinity than the reference antibody 280.346.TSY (0.69 nM)

Because of the potential avidity effect observed when a bivalentmolecule is used as a ligand during affinity measurement, a reverseexperimental setup was tested. In this set-up, the antibodies werecoated to 250 RU using standard methods. Monomeric human IL22R was addedat different concentrations, and the affinities calculated using theBIAevaluation software using a Langmuir 1:1 fitting are shown in Table21 below.

TABLE 21 Binding affinity of 230C9 to human IL-22R ka (1/Ms) kd (1/s) KD(M) 230C9 hIL22R 9.2E+05 2.2E−04 2.2E−10

Unfortunately the antibody 223G5 was inactivated by the coating processand no affinity could be determined using this setup.

The affinity of 230C9 is consistent independent of the setup of theaffinity measurement, showing that the low coating used in bothconditions is sufficient to reduce any avidity effect.

12.2 Inhibition of IL-22 and IL-20 Dependent IL-22R1 Activation andSignalling

Antibodies 218A7 (not germlined), and 230C9 (the germlined equivalent)were tested alongside other germlined antibodies for their ability toneutralize IL-22 and IL-20-mediated signalling via IL-22R1. Testing wasperformed using the cell-based proliferation assays described inExamples 6 and 9.

FIG. 9A shows the ability of 218A7 and 230C9 to neutralizeIL-22-dependent activation of IL-22R1. FIG. 9B shows the ability ofdifferent batches of 230C9 to neutralize IL-20-dependent activation ofIL-22R1. The relative potencies of the antibodies tested are shown inTables 22 and 23 below.

TABLE 22 Potency of the 218A7 and 230C9 antibodies in blocking IL-22signalling via IL22R (BWhIL22R cell line) IC50 Relative blocking potencyto IL-22 (nM) 280.346.TSY 218A7 0.28 2.5 230C9 0.56 1.2 230D5 0.62 1.1230H2 0.66 1.0 280.346.TSY 0.69 1 230B7 0.73 0.95

TABLE 23 Potency of the 230C9 antibody in blocking IL-20 signalling viaIL22R (Baf3hIL22R/IL20Rb cell line) IC50 blocking Relative potency toIL-20 (nM) 280.346.TSY 280.346.TSY 1.487 230C9-N297Q (#1)* 0.7807 1.9230C9-N297Q (#2)* 0.5671 2.6 *Different purification batches of the230C9 antibody12.3 Confirmatory Epitope Mapping

Confirmatory epitope mapping for germlined antibodies 230C9 and 223G5was carried out using FACS analysis, as described in Example 8.2.Antibody 230C9 was found to compete for binding to IL-22R1 with thebenchmark antibody 280.346.TSY and also 223G5. Antibody 223G5 did notcompete for binding to IL-22R1 with the benchmark antibody 280.346.TSY.

The results of the FACS analysis were combined with the results of theproliferation assays described above and the conclusions regardingepitopes are shown in FIG. 10. As indicated, it can be concluded that(i) antibody 230C9 binds to an epitope distinct but overlapping ascompared with the epitopes bound by 280.346.TSY; and (ii) antibody 223G5binds to an epitope distinct from 280.346.TSY.

FACS analysis was also carried out as described in Example 8.2 to studythe binding of the germlined antibodies to various IL-22R mutants. Theresults are shown in Table 24 below.

TABLE 24 Antibody binding to cells expressing IL22R mutants BW-hIL-22Rmutants A209D W208A Y60A R112A 280.346.TSY + + X X 230C9 + + + +/X 223G5X + + + +/− = partial recognition of mutant having R112A

These results confirm that antibodies 230C9 and 223G5 bind to an epitopethat does not include Y60. The mutation A209D was found to affectbinding of 223G5 whereas binding of llama antibody 166G8 was unaffectedby this change. This is likely attributable to difficulties in detectingthe sensitivity of 166G8 to the A209D mutation (166G8 was tested onlyonce), and possibly attributable to slight changes in the CDR sequencesduring germlining of 166G8. These data confirm that 280.346.TSY binds tothe D1 of IL22R, that 223G5 binds to the D2 of IL22R and that 230C9 hasan overlapping but distinct epitope.

12.4 Species Cross-Reactivity of Germlined Antibodies

Antibody 230C9 binds human IL-22R but exhibits no cross-reactivity forIL-22R from mouse, rat and rabbit. This is consistent with 230C9 havinga distinct epitope as compared with the benchmark antibody280.346.TSY—see 12.3 above.

The capacity of the antibodies to bind cyno-IL22R once expressed oncells was tested by making chimeric constructs of the IL22Rextracellular domain (ECD) from the different species fused to the humantransmembrane (TM) and human intracellular domains (ID). Although theefficiency of the transient transfection was not large, FACS analysisshowed that the 230C9-N297Q antibody, but not the 223G5-N297Q bound tothe cynoIL22R_ECD-humanTM_humanICD as observed by the presence of abroader peak on the FACS plot (see FIG. 11 with the arrows indicatingthe broader peaks).

Furthermore, when the chimeric constructs were also co-transfected witha STAT3-reporter gene into HEK cells, the activity of the IL22R afteraddition of human IL22 could be measured in the presence or absence ofneutralizing antibody. The results presented in Table 25 below show thatall the antibodies tested were able to neutralize the human IL22R andthat only the primate cross-reactive (230C9 and 280.346.TSY) were ableto bind and block the activity of the cyno and rhesus chimerassuggesting that these antibodies will be functional in primate studies.

TABLE 25 Cross-reactivity of IL-22R antibodies with human, rhesus andcyno IL-22R EC50 (nM) EC50 (nM) EC50 (nM) Human ECD Rhesus ECD Cyno ECD224C4 0.30 No effect No effect 230C9 0.28 0.48 0.57 280.346.TSY 0.260.43 0.1612.5 Immunogenicity Analysis

The immunogenicity of the germlined antibodies was assessed usingprediction tools. In particular, the presence of potential immunogenicpeptides in the variable domains was assessed using the Ionza's Epibase™platform (DRB-1 score) using the “HLA class II-Caucasian v3.0″settings”. This platform analyses the HLA binding specificities of all10-mer peptides derived from the VH and VL sequences. Profiling was doneat the allotype level for 15 DRB1, 6 DRB3/4/5, 12 DQ and 7 DP, i.e. 40HLA class II receptors in total. Strong and medium binders of DRB1,DRB3/4/5 were identified, as well as the strong binders of DQ and DPepitopes. Epitope counting was done separately for strong and mediumaffinity DRB1 binders. Peptides binding to multiple allotypes of thesame group were counted as one. An approximate score expressing aworst-case immunogenic risk was calculated as follows: Score=Σ(epitopecount×allotype frequency). In other words, the number of epitopesaffecting a particular HLA allotype is multiplied by the allelefrequency of the affected allotype. For a given sequence, the productswere summed for all DRB1 allotypes used in the study that are present in2% or more of the Caucasian population.

The results are shown in Table 26 below.

TABLE 26 DRB1 scores for the germlined IL-22R antibodies as comparedwith various other commercial antibodies DRB1 Score (VH + VL) Source230C9 555 Germlined Ilama 223G5 936 Germlined Ilama Adalimumab 830 Human(phage display) Trastuzumab (Herceptin) 959 Humanized Palivizumab(Synagis) 985 Humanized Dupilumab 1124 Human (Velocimmune) Alemtuzumab(Campath) 1409 Humanized Rituximab (Rituxan) 1769 Chimeric Infliximab(Remicade) 1873 Chimeric Mouse antibody 3832 Mouse

As can be seen from Table 26, antibody 230C9 has a very low DRB1 score,well below other commercial antibodies whilst 223G5 has a low DRB1 scorecomparable to other commercially available human or humanizedantibodies. These data suggest that 230C9 (and 223G5) will have apreferable immunogenicity profile.

Example 13 Pharmacokinetic (PK) Study in Cynomolgus Monkeys of Antibody230C9-N297Q

Pharmacokinetic analysis of antibody clone 230C9 was performed. TwoCynomolgus monkeys were injected intravenously with a single 10 mg/kgdose of antibody for 2 h. Samples were taken at different time pointsand tested for plasma concentration of mAb by ELISA. Specifically, amicrotiterplate (Maxisorb Nunc) was coated with 2 μg/ml recombinanthIL22R (Biotechne; cat 2770-LR) in PBS overnight at 4° C. The plate waswashed 3 times with PBS-Tween and blocked for 2 hours with 250 μl PBS-1%casein. After 3 washes with PBS-Tween, the samples were applied. Alldilutions were made in 1% pooled plasma (this is a pool from 3 naivecynomolgus monkeys). The samples were allowed to bind for 2 hours at RT.Plates were then washed 5 times with PBS-Tween and goat biotinylatedanti-human IgG heavy and light chain monkey adsorbed polyclonalantibodies coupled to HRP were applied at a 50,000-fold dilution(Bethyl, catno: A80-319P) and allowed to bind for 1 hour at RT. Plateswere then washed 5 times with PBS-Tween and s(HS)TMB weakener (SDT, #sTMB-W) was added. The staining was allowed to proceed for 10 minutesand then stopped with 1N H₂SO, after which the Optical Density wasmeasured at 450 nm. The samples were analysed 3 times and 230C9-N297Q(from the same batch that was injected into the animals) was used for astandard curve.

The relevant PK parameters for a 2-compartment IV-infusion modelanalysis are shown in Table 27. The pharmacokinetic profiles for230C9-N297Q antibody is shown graphically in FIG. 12 (the result shownis the average result of the two monkeys). This data clearly show thatthe antibody has a long mean residency time (MRT). Interestingly,although the antibody contains a non-glycosylated IgG1 Fc region withoutany modification to improve the half-life, the half-life of 230C9-N297Qis surprisingly long. Specifically, 230C9-N297Q has a half-life of about19.4 days. Thus, the extended half-life of the antibodies of theinvention appears to be due to the properties of the Fab regions.

TABLE 27 PK parameters T½ MRT V1 V2 CL K21 Cmax C at day 30 19.4 26.2 4568 4.3 0.35 216 ~26 days days ml/kg ml/kg ml/day μg/ml μg/ml

A further PK study was carried out in cynomolgus monkeys, byadministering single IV doses (0.1, 1, 3, 10 and 30 mg/kg) of230C9-N297Q to 10 monkeys in total. The kinetics in cynomolgus monkeysdisplayed two-compartment non-linear kinetics. At doses lower than 10mg/kg, clearance (CL) increased with lowering of the dose resulting in amore than dose proportional decrease in exposure (AUC) with decreasingdoses. This is a well-known phenomenon for monoclonal antibodies (seeTable 28 and FIGS. 13A-13B).

TABLE 28 PK parameters based on 2 monkeys/per dose and 5 dose groups.Parameter Estimate Units Stderr CV % tvV 39.7 mL/kg 2.77 7.0 tvCl 3.60mL/(kg * day) 0.38 10.5 tvV2 27.7 mL/kg 5.40 19.5 tvCl2 42.1 mL/(kg *day) 24.3 57.8 tvVmax 24.9 μg/(kg * day) 7.41 29.8 tvKm 1.0 μg/mL 0.019.96

Scaling of these parameters in order to predict human pharmacokineticsresults in a human profile similar to what can be expected based onempiric findings from other human IgG1 molecules, including a pointestimate of human half-life (T½) of about 18 days in agreement with theprevious study reported above.

Example 14 Pharmacodynamic (PD) Effects of Antibody 230C9 N297Q in aCynomolgus Monkey

The pharmacodynamic effect of antibody 230C9 N297Q (ARGX-112) wasanalysed in a cynomolgus monkey by administering to the monkey by IVinjection 230C9 at different doses (0.3, 1, 3, 10, 30 mg/kg). A skinsection of the monkey was also treated with imiquimod (IMQ) to assessthe effects of 230C9 N297Q on skin inflammation. IMQ has been reportedto induce skin inflammation in mice (Van Belle et al. 2012, J Immunol.January 1; 188(1):462-9), and one report also demonstrates similareffects in non-human primate (Poirier et al, 2016, Exp Dermatol. March;25(3):233-4; Poirier et al, 2016, J Immunol. January 1; 196(1):274-83),and in human (Vinter et al, 2015, Br J Dermatol. February;172(2):345-53).

Following 5 days of IMQ treatment, a biopsy was taken from a non-IMQtreated area of skin and the IMQ-treated area of skin to assess theeffects of 230C9 N297Q on the IMQ-induced effects. The effects wereassessed by comparing epidermal thickness and frequency of proliferatingnuclei in the epidermis (Ki67 frequency), and the results are shown inFIGS. 14A-14B. Increasing doses of antibody 230C9 N297Q were able tonormalize epidermal thickness (FIG. 14A) and reduce the frequency ofKi67 positive nuclei (FIG. 14B). The EC₅₀ was approximately 3 mg/kg.

Example 15 Serum PK and Skin Explant PD Response Relationship after aSingle IV Infusion of 230C9 N297Q in Cynomolgus Monkeys

Skin target engagement was investigated in cynomolgus monkeysadministered a single 15-min intravenous infusion (5 ml/kg) of antibody230C9 N297Q. Female monkeys were left either untreated or exposed to 1,5 or 30 mg/kg 230C9 on test day 1 (three animals per group). Blood wascollected from all dosed animals (3×150 μL) at different time pointswith the last sample being withdrawn at day 7. Serum samples (1% v/v)were tested for 230C9 N297Q exposure levels by ELISA.

Skin punch biopsies (3 mm) were sampled at day −1 (pre-dose) and day 7from each animal and incubated in the presence (day −1, day 7) orabsence (day 7) of rhIL-22 for 24 h at 37° C. in humidified air/CO₂(95%/5%) before measuring FLG2 mRNA levels by qPCR. The results areshown in FIG. 15.

Stimulation by rhIL22 resulted in a 5-fold reduction in total skin FLG2mRNA levels. IL-22-mediated FLG2 transcript repression was significantlyrestored in 230C9 N297Q-treated animals indicating significant skinexposure in all dose groups.

Example 16 Effect of 230C9 N297Q in Viable Human and Cynomolgus MonkeyEx Vivo Skin Explants

Freshly sourced abdominal skin from healthy human donors was used toassess the ability of antibody 230C9 N297Q to inhibit rhIL-22 inducedDEFB4 mRNA levels.

Skin punch biopsies (3 mm) were placed in an upright position in 96-welltissue plates and cultured in air liquid interface in 100 μL EpiLifemedium supplemented with Human Keratinocyte Growth Supplement (HKGS)without hydrocortisone (Invitrogen).

The samples were incubated with increasing concentrations of antibody230C9 N297Q for 24 h at 37° C. in humidified air/CO₂ (95%/5%) prior tostimulation with 20 ng/ml rhIL-22 (R&D Systems) for an additional 24 hat 37° C. The relative DEFB4 gene expression levels in skin lysates weredetermined by real-time quantitative PCR (qPCR) using validated geneexpression assays (Applied Biosystems) and an ABI PRISM® 7900HT sequencedetection system.

FIG. 16 shows a dose dependent reduction of rhIL-22 induced DEFB4 mRNAlevels from one representative experiment (EC₅₀ [C195%]=4 nM [0.5-29nM]; 4 donors). A similar potency was obtained if increasing the 230C9N297Q pre-incubation time to 48 h suggesting IL-22RA binding equilibriumhas been reached. An isotype control antibody had no effect on rhIL-22mediated increments of DEFB4 mRNA levels.

In freshly isolated skin biopsies (3 mm) from cynomolgus monkeys (Macacafascicularis) the DEFB4 gene was not regulated by rhIL-22. In contrast,both FLG2 and LOR were down-regulated following 24 h incubation withrhIL-22 using the same protocol as described for the human skin explantset-up. These responses were completely blocked by 24 h pre-incubationwith antibody 230C9 N297Q (EC₅₀=11 nM; both genes) as shown in FIGS.17A-17B.

Example 17 Efficacy of 230C9 N297Q in Human Keratinocytes

The assay was designed to test the potency of IL-22R antibodies in afunctional assay in primary human keratinocytes. The cells werestimulated with a cytokine mixture of IL-4, IL-13, IL-22 (all 10 ng/mL)and IFN-γ, 1 ng/mL for 48 hours and then the level of CCL2 was measuredin the culture supernatant using the MSD platform.

The keratinocytes were suspended in EpiLife medium (Life Technologies)with the following growth supplements added: EGF; BPE; Insulin;Transferrin and Gentamicin/Amphotericin. Cells were seeded in 384 wellwhite proxy plates (Perkin Elmer) and incubated for 2 hours at 37° C.,5% CO₂/95% air. The cells were then treated with 80 nl antibody (atdifferent concentrations as shown in FIG. 18) or vehicle. Then, 40 μlstimulation mixture (IL-4, IL-13, IL-22 (R&D systems, all 10 ng/mL finalconcentration) and IFN-γ (R&D systems, 1 ng/mL final concentration)) wasadded and the plates were incubated at 37° C., 5% CO₂/95% air for 2days. Control wells were treated with a mixture of IL-4 and IL-13 (both10 ng/mL) and IFN-γ, 1 ng/mL, and defined 100% inhibition of IL-22signalling. The concentration of CCL2 in the culture supernatant wasmeasured with MSD CCL2 kit (Mesoscale Cat # K151AYB-2). The viability ofthe cells was measured by PrestoBlue® reagent.

The potency of 230C9 N297Q was tested in 5 experiments and showeddose-dependent inhibition of CCL2 levels with EC₅₀ values of 0.10 nM[0.042-0.24 nM]. Representative experimental results from one experimentare shown in FIG. 18.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingfigures. Such modifications are intended to fall within the scope of theappended claims. Moreover, all embodiments described herein areconsidered to be broadly applicable and combinable with any and allother consistent embodiments, as appropriate.

The invention claimed is:
 1. An antibody, or an antigen-binding fragmentthereof, which binds to human interleukin 22 receptor (IL-22R) protein,wherein the antibody or antigen-binding fragment comprises a heavy chainvariable domain (VH) and a light chain variable domain (VL), the VHcomprising a combination of variable heavy chain CDR sequences: HCDR3comprising SEQ ID NO: 6; HCDR2 comprising SEQ ID NO: 36; and HCDR1comprising SEQ ID NO: 34, and the VL comprising combination of variablelight chain CDR sequences: LCDR3 comprising SEQ ID NO: 54; LCDR2comprising SEQ ID NO: 47; and LCDR1 comprising SEQ ID NO:
 16. 2. Theantibody or antigen-binding fragment of claim 1, wherein the VH domainand/or the VL domain is a humanised or germlined variant of acamelid-derived VH or VL domain.
 3. The antibody or antigen-bindingfragment of claim 1 which contains the hinge region, CH2 domain and/orCH3 domain of a human IgG.
 4. A pharmaceutical composition comprisingthe antibody or antigen-binding fragment according to claim 1 and atleast one pharmaceutically acceptable carrier or excipient.
 5. Theantibody or antigen-binding fragment of claim 1, wherein the VHcomprises an amino acid sequence at least 90% identical to the aminoacid sequence of SEQ ID NO:
 63. 6. The antibody or antigen-bindingfragment of claim 1, wherein the VH comprises the amino acid sequence ofSEQ ID NO:
 63. 7. The antibody or antigen-binding fragment of claim 1,wherein the VL comprises an amino acid sequence at least 90% identicalto the amino acid sequence of SEQ ID NO:
 64. 8. The antibody orantigen-binding fragment of claim 1, wherein the VL comprises the aminoacid sequence of SEQ ID NO:
 64. 9. An antibody or antigen-bindingfragment thereof, which binds to human interleukin 22 receptor (IL-22R)protein, wherein the antibody or antigen-binding fragment comprises aheavy chain and a light chain, wherein the heavy chain comprises theamino acid sequence of SEQ ID NO: 67 and wherein the light chaincomprises the amino acid sequence of SEQ ID NO:
 68. 10. A pharmaceuticalcomposition comprising the antibody or antigen-binding fragmentaccording to claim 9 and at least one pharmaceutically acceptablecarrier or excipient.
 11. An antibody which binds to human interleukin22 receptor (IL-22R) protein, wherein the antibody comprises a heavychain comprising a heavy chain variable domain (VH) and a light chaincomprising a light chain variable domain (VL), the VH comprising theamino acid sequence of SEQ ID NO: 63 and the VL comprising the aminoacid sequence of SEQ ID NO:
 64. 12. The antibody of claim 11, furthercomprising the hinge region, CH2 domain and/or CH3 domain of a humanIgG1 antibody.
 13. The antibody of claim 11, wherein the heavy chaincomprises an amino acid sequence at least 90% identical to the aminoacid sequence of SEQ ID NO:
 67. 14. The antibody of claim 11, whereinthe heavy chain comprises an amino acid sequence at least 95% identicalto the amino acid sequence of SEQ ID NO:
 67. 15. The antibody of claim11, wherein the heavy chain comprises an amino acid sequence at least98% identical to the amino acid sequence of SEQ ID NO:
 67. 16. Theantibody of claim 11, wherein the heavy chain comprises an amino acidsequence at least 99% identical to the amino acid sequence of SEQ ID NO:67.
 17. The antibody of claim 11, wherein the heavy chain comprises theamino acid sequence of SEQ ID NO:
 67. 18. The antibody of claim 11,wherein the light chain comprises an amino acid sequence at least 90%identical to the amino acid sequence of SEQ ID NO:
 68. 19. The antibodyof claim 11, wherein the light chain comprises an amino acid sequence atleast 95% identical to the amino acid sequence of SEQ ID NO:
 68. 20. Theantibody of claim 11, wherein the light chain comprises an amino acidsequence at least 98% identical to the amino acid sequence of SEQ ID NO:68.
 21. The antibody of claim 11, wherein the light chain comprises theamino acid sequence of SEQ ID NO:
 68. 22. The antibody of claim 11,wherein the heavy chain comprises an amino acid sequence at least 95%identical to the amino acid sequence of SEQ ID NO: 67 and wherein thelight chain comprises an amino acid sequence at least 95% identical tothe amino acid sequence of SEQ ID NO:
 68. 23. A pharmaceuticalcomposition comprising the antibody or antigen-binding fragmentaccording to claim 11 and at least one pharmaceutically acceptablecarrier or excipient.